U.S. patent application number 10/128990 was filed with the patent office on 2004-05-13 for substituted tetracycline compounds for the treatment of malaria.
Invention is credited to Draper, Michael, Nelson, Mark L..
Application Number | 20040092490 10/128990 |
Document ID | / |
Family ID | 23097495 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040092490 |
Kind Code |
A1 |
Draper, Michael ; et
al. |
May 13, 2004 |
Substituted tetracycline compounds for the treatment of malaria
Abstract
This invention provides a method for treating or preventing
malaria in a subject. The method includes administering to the
subject an effective amount of a substituted tetracycline compound,
such that malaria is treated or prevented. In one aspect, the
invention relates to pharmaceutical compositions which include an
effective amount of a tetracycline compound to treat malaria in a
subject and a pharmaceutically acceptable carrier. The substituted
tetracycline compounds of the invention can be used to in
combination with one or more anti-malarial compounds or can be used
to treat or prevent malaria which is resistant to one or more other
anti-malarial compounds.
Inventors: |
Draper, Michael; (Plaistow,
NH) ; Nelson, Mark L.; (Wellesley, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
23097495 |
Appl. No.: |
10/128990 |
Filed: |
April 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60286193 |
Apr 24, 2001 |
|
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|
Current U.S.
Class: |
514/152 ;
514/284; 514/434; 514/453 |
Current CPC
Class: |
Y02A 50/411 20180101;
A61K 31/385 20130101; Y02A 50/473 20180101; A61K 31/353 20130101;
A61P 33/06 20180101; A61P 31/04 20180101; Y02A 50/30 20180101; A61K
31/473 20130101; A61K 31/65 20130101; A61K 31/353 20130101; A61K
2300/00 20130101; A61K 31/385 20130101; A61K 2300/00 20130101; A61K
31/473 20130101; A61K 2300/00 20130101; A61K 31/65 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/152 ;
514/284; 514/434; 514/453 |
International
Class: |
A61K 031/65; A61K
031/473; A61K 031/353; A61K 031/385 |
Claims
1. A method for treating or preventing malaria in a subject,
comprising administering to said subject an effective amount of a
substituted tetracycline compound, such that malaria is treated in
said subject.
2. The method of claim 1, wherein said tetracycline compound is of
formula I: 286wherein: X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S,
NR.sup.6, or O; R.sup.2, R.sup.2', R.sup.4', and R.sup.4" are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety; R.sup.4 is
NR.sup.4'R.sup.4", alkyl, alkenyl, alkynyl, hydroxyl, halogen, or
hydrogen; R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety; R.sup.10 is hydrogen, a prodrug moiety, or linked
to R.sup.9 to form a ring; R.sup.5 is hydroxyl, hydrogen, thiol,
alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,
alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;
R.sup.6 and R.sup.6' are independently hydrogen, methylene, absent,
hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; R.sup.7 is hydrogen, alkylamino, dialkylamino, or a
malaria interacting moiety; R.sup.9 is hydrogen, or a malaria
interacting moiety; R.sup.8 is hydrogen, hydroxyl, halogen, thiol,
alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; R.sup.13 is hydrogen,
hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; Y' and Y are each
independently hydrogen, halogen, hydroxyl, cyano, sulfhydryl,
amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; with the proviso that
the compound of formula I is not oxytetracycline, demeclocycline,
doxycycline, chlorotetracycline, minocycline, or tetracycline; and
pharmaceutically acceptable salts thereof.
3. The method of claim 2, wherein R.sup.2, R.sup.2', R.sup.3,
R.sup.8, R.sup.10, R.sup.11, and R.sup.12 are hydrogen; R.sup.4 is
NR.sup.4'R.sup.4"; R.sup.4' and R.sup.4" are alkyl, and X is
CR.sup.6R.sup.6'.
4. The method of claim 3, wherein R.sup.5, R.sup.6, and R.sup.6'
are hydrogen, and R.sup.7 is dimethylamino.
5. The method of claim 3, wherein R.sup.5 is hydroxy or a prodrug
moiety, R.sup.6 is methyl, R.sup.6' is hydrogen and R.sup.7 is
hydrogen.
6. The method of claim 4 or 5, wherein R.sup.9 is a malaria
interacting moiety.
7. The method of claim 6, wherein said malaria interacting moiety
comprises a substituted or unsubstituted aryl group.
8. The method of claim 7, wherein said malaria interacting moiety
is substituted phenyl.
9. The method of claim 8, wherein said malaria interacting moiety
is substituted with alkoxy, alkyl, alkenyl, alkynyl, aryl, amino,
cyano, hydroxy, nitro, or a halogen.
10. The method of claim 9, wherein said malaria interacting moiety
is methylene dioxyphenyl.
11. The method of claim 9, wherein said aryl group is substituted
with an alkyl.
12. The method of claim 11, wherein said alkyl is substituted with
a heterocycle.
13. The method of claim 4 or 5, wherein said malaria interacting
moiety is substituted or unsubstituted alkenyl or alkynyl.
14. The method of claim 4 or 5, wherein said malaria interacting
moiety is --NR.sup.9cC(.dbd.Z')ZR.sup.9a.
15. The method of claim 14, wherein Z is N and Z' is O.
16. The method of claim 14 or 15, wherein R.sup.9a is aryl.
17. The method of claim 14, wherein Z is O, Z' is O, and R.sup.9a
is alkyl.
18. The method of claim 3, wherein R.sup.6 and R.sup.6' are
hydrogen, and R.sup.5 is a prodrug moiety or hydrogen.
19. The method of claim 18, wherein R.sup.7 is a malaria
interacting moiety.
20. The method of claim 19, wherein R.sup.7 contains 4 to 20 atoms,
selected from the group consisting of carbon, nitrogen, sulfur, and
oxygen.
21. The method of claim 19, wherein said malaria interacting moiety
comprises an aryl group.
22. The method of claim 20, wherein said aryl group is substituted
or unsubstituted phenyl.
23. The method of claim 22, wherein said phenyl group is
substituted with halogen, alkoxy, amino, acyl, alkyl, nitro,
formyl, amido, alkyl, alkenyl, alkynyl, or aryl.
24. The method of claim 23, wherein said alkoxy group is methoxy,
ethoxy, propoxy, methylene dioxy, or ethylene dioxy.
25. The method of claim 23, where said alkyl group is substituted
or substituted methyl, ethyl, propyl, butyl or pentyl.
26. The method of claim 25, wherein said alkyl group is substituted
with an amino, carbocyclic or heterocyclic group.
27. The method of claim 23, wherein said acyl group is acetyl.
28. The method of claim 21, wherein said aryl group is substituted
or unsubstituted heteroaryl.
29. The method of claim 28, wherein said heteroaryl is thienyl,
imidazolyl, pyrolyl, pyridinyl, furanyl, pyrimidinyl, or
benzofuranyl.
30. The method of claim 19 or 20, wherein said malaria interacting
moiety is substituted or unsubstituted alkynyl.
31. The method of claim 30, wherein said alkyl is substituted with
a substituted or unsubstituted aryl group.
32. The method of claim 19, wherein said malaria interacting moiety
is alkyl or alkenyl.
33. The method of claim 32, wherein said malaria interacting moiety
is C.sub.1-C.sub.15.
34. The method of any one of claims 3-33, wherein R.sup.5 is an
alkyl ester.
35. The method of any one of claims 3-33, wherein R.sup.5 is
hydroxy.
36. The method of any one of claims 19-35, wherein R.sup.9 is
hydrogen.
37. The method of any one of claims 19-35, wherein R.sup.9 is a
malaria interacting moiety.
38. The method of claim 2, wherein said compound is selected from
the group consisting of: 287288289290291292
39. The method of claim 2, wherein said compound is selected from
the group consisting of: 293294295296297298299300
40. The method of claim 2, wherein said compound is a compound
shown in Table 1.
41. The method of any one of claims 1-40, wherein said subject is a
human.
42. The method of anyone of claims 1-41, wherein said substituted
tetracycline compound is has anti-microbial gram positive
activity.
43. The method of claim 42, wherein said anti-microbial gram
positive activity is greater than about 0.05 .mu.g/ml.
44. The method of claim 43, wherein said anti-microbial gram
positive activity is greater than about 5 .mu.g/ml.
45. The method of any one of claims 1-44, wherein said substituted
tetracycline compound has a cytotoxicity of 25 .mu.g/ml or
greater.
46. The method of any one of claims 1-45, wherein said substituted
tetracycline compound has a MIC of 150 nM or less.
47. The method of claim 46, wherein said substituted tetracycline
compound has a MIC of 50 nM or less.
48. The method of claim 47, wherein said substituted tetracycline
compound has a MIC of 10 nM or less.
49. The method of claim 48, wherein said substituted tetracycline
compound has an MIC or 5 nM or less.
50. The method of any one of claims 1-49, wherein said malaria is
caused by a plasmodium protozoan selected from the group consisting
of: P. falciparum, P. vivax, P. ovale, and P. malariae.
51. The method of any one of claims 1-50, wherein said malaria is
resistant to one or more anti-malarial compounds selected from the
group consisting of: proguanil, chlorproguanil, trimethoprim,
chloroquine, mefloquine, lumefantrine, atovaquone,
pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine,
quinine, quinidine, amodiaquine, amopyroquine, sulphonamides,
artemisinin, arteflene, artemether, artesunate, primaquine, and
pyronaridine.
52. The method of any one of claims 1-51, wherein said malaria is
resistant to one or more anti-malarial compounds selected from the
group consisting of: proguanil, chlorproguanil, pyrimethamine,
chlorquine, mefloquine, halofantrine, quinine, and quinidine.
53. The method of any one of claims 1-52, further comprising
administering a supplementary compound.
54. The method of claim 53, wherein said supplementary compound
treats a symptom selected from the group consisting of: headache,
malaise, anemia, splenomegaly, and fever.
55. The method of claim 53, wherein said supplementary compound is
an anti-malarial compound.
56. The method of claim 55, wherein said anti-malarial compound is
selected from the group consisting of: proguanil, chlorproguanil,
trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone,
pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine,
quinine, quinidine, amodiaquine, amopyroquine, sulphonamides,
artemisinin, arteflene, artemether, artesunate, primaquine,
pyronaridine, proguanil, chloroquine, mefloquine,
pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine,
quinine, proguanil, chloroquine, mefloquine,
1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide, and
combinations thereof.
57. A method for preventing malaria in a mammal, comprising
administering to said mammal an effective amount of a substituted
tetracycline compound, such that malaria is prevented in said
mammal, wherein said tetracycline compound is of formula I:
301wherein: X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6,
or O; R.sup.2, R.sup.2', R.sup.4', and R.sup.4" are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety; R.sup.4 is
NR.sup.4'R.sup.4", alkyl, alkenyl, alkynyl, hydroxyl, halogen, or
hydrogen; R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety; R.sup.10 is hydrogen, a prodrug moiety, or linked
to R.sup.9 to form a ring; R.sup.5 is hydroxyl, hydrogen, thiol,
alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,
alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;
R.sup.6 and R.sup.6' are independently hydrogen, methylene, absent,
hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; R.sup.7 is hydrogen, alkylamino, dialkylamino, or a
malaria interacting moiety; R.sup.9 is hydrogen, or a malaria
interacting moiety; R.sup.8 is hydrogen, hydroxyl, halogen, thiol,
alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; R.sup.13 is hydrogen,
hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; Y' and Y are each
independently hydrogen, halogen, hydroxyl, cyano, sulfhydryl,
amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; with the proviso that
the compound of formula I is not oxytetracycline, demeclocycline,
doxycycline, chlorotetracycline, minocycline, or tetracycline; and
pharmaceutically acceptable salts thereof.
58. The method of claim 57, wherein said substituted tetracycline
compound is selected from the group consisting of:
302303304305306307308309310311- 312313314
59. The method of claim 57, wherein said substituted tetracycline
compound is a compound shown in Table 1.
60. The method of any one of claims 57-59, wherein said substituted
tetracycline compound is has anti-microbial gram positive
activity.
61. The method of claim 60, wherein said anti-microbial gram
positive activity is greater than about 0.05 .mu.g/ml.
62. The method of claim 61, wherein said anti-microbial gram
positive activity is greater than about 5 .mu.g/ml.
63. The method of anyone of claims 57-62, wherein said substituted
tetracycline compound has a cytotoxicity of 25 .mu.g/ml or
greater.
64. The method of any one of claims 57-63, wherein said substituted
tetracycline compound has a MIC of 150 nM or less.
65. The method of claim 64, wherein said substituted tetracycline
compound has a MIC of 50 nM or less.
66. The method of claim 65, wherein said substituted tetracycline
compound has a MIC of 10 nM or less.
67. The method of claim 66, wherein said substituted tetracycline
compound has an MIC or 5 nM or less.
68. A pharmaceutical composition comprising an effective amount of
a substituted tetracycline compound to treat malaria in a mammal
and a pharmaceutically acceptable carrier, wherein said
tetracycline compound is of formula I: 315wherein: X is
CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or O; R.sup.2,
R.sup.2', R.sup.4', and R.sup.4" are each independently hydrogen,
alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,
heteroaromatic or a prodrug moiety; R.sup.4 is NR.sup.4'R.sup.4",
alkyl, alkenyl, alkynyl, hydroxyl, halogen, or hydrogen; R.sup.3,
R.sup.11 and R.sup.12 are each hydrogen, or a pro-drug moiety;
R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9 to
form a ring; R.sup.5 is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,
alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
alkyl carbonyloxy, or aryl carbonyloxy; R.sup.6 and R.sup.6' are
independently hydrogen, methylene, absent, hydroxyl, halogen,
thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R.sup.7
is hydrogen, alkylamino, dialkylamino, or a malaria interacting
moiety; R.sup.9 is hydrogen, or a malaria interacting moiety;
R.sup.8 is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl,
alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, or an arylalkyl; R.sup.13 is hydrogen, hydroxy, alkyl,
alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, or an arylalkyl; Y' and Y are each independently
hydrogen, halogen, hydroxyl, cyano, sulfhydryl, amino, alkyl,
alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, or an arylalkyl; with the proviso that the compound of
formula I is not oxytetracycline, demeclocycline, doxycycline,
chlorotetracycline, minocycline, or tetracycline; and
pharmaceutically acceptable salts thereof.
69. The pharmaceutical composition of claim 68, wherein said
substituted tetracycline compound is selected from the group
consisting of: 316317318319320321322323324325326327328
70. The method of claim 68, wherein said substituted tetracycline
compound is a compound shown in Table 1.
71. The pharmaceutical composition of any one of claims 68-70,
further comprising a supplementary anti-malarial compound.
72. The pharmaceutical composition of claim 71, wherein the
supplementary anti-malarial compound selected from the group
consisting of proguanil, chlorproguanil, trimethoprim, chloroquine,
mefloquine, lumefantrine, atovaquone, pyrimethamine-sulfadoxine,
pyrimethamine-dapsone, halofantrine, quinine, quinidine,
amodiaquine, amopyroquine, sulphonamides, artemisinin, arteflene,
artemether, artesunate, primaquine,
1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide and
pyronaridine.
73. A packaged malarial treatment or prophylactic, comprising a
substituted tetracycline compound packaged with instructions for
using an effective amount of the tetracycline compound to treat or
prevent malaria.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Serial No. 60/286,193, entitled "Substituted
Tetracycline Compounds for the Treatment of Malaria," filed on Apr.
24, 2001, the entire contents of which are hereby incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Every year there are about 300-500 million clinical cases of
malaria. About 40% of the world's population is at risk of
acquiring the disease. (Croft (2000) BMJ 321:154-160.) Malaria is
characterized by headache, malaise, anemia, splenomegaly, and
paroxysms with cold, hot, and wet the stages. (Winstanley (1998)
Journal of the Royal College of Physicians of London
32(3):203-207.) Hemolysis and ischemia cause the majority of the
symptoms seen with acute malaria. Malaria is caused by protozoa of
the genus Plasmodium. There are over 100 species of which 22 infect
nonhuman primates and 82 are pathogenic for reptiles and birds. The
four species that commonly infect man are: P. falciparum, P.
malariae, P. vivax, and P ovale. Malaria may be transmitted by a
bite of the Anopheles mosquito, infected blood transfusions,
transplacentally, and in laboratory inoculation accidents.
[0003] Plasmodia have a complex life cycle where the sexual phase
occurs in the Anopheles mosquito and the asexual phase takes place
in the veterbrate host (i.e. a human). (Randall, et al. (1985)
Pediatric Clinics of North America 32(4):893-916.) The process of
sexual reproduction in the mosquito is called Sporogony and
includes the period from gametocyte maturation to sporozoite
development. When a female Anopheles mosquito feeds, it takes up
gametocytes present in the blood of an infected host. The
gametocytes taken up by the mosquito pass to the mosquito's gut. A
zygote is formed by the fusion of the microgamete and macrogamete.
After 12 to 24 hours, the zygotes elongates and becomes motile and
is called an ookinete. The ookinete later penetrates the mosquito's
stomach to form an oocyst which divides into thousands of
spindle-shaped sporozoites which are released throughout the
mosquito's body.
[0004] When a blood meal is taken by an infected Anopheles
mosquito, sporozoites from the salivary glands of the mosquito are
inoculated into the bloodstream of the veterbrate host (i.e. human)
and are carried to the liver. At the end of the hepatic phase of
development, thousands of merozoites are released into the
circulation where they bind to and enter red blood cells. The
erythrocyte phase of asexual reproduction is termed Schizogeny.
When the infected erythrocytes rupture, they release merozoites
which can invade more red blood cells. Other released merozoites
become gametophytes capable of infecting feeding mosquitoes and
restarting the life cycle of the Plasmodia.
SUMMARY OF THE INVENTION
[0005] This invention pertains, at least in part, to a method for
treating or preventing malaria in a subject by administering an
effective amount of a substituted tetracycline compound. The method
includes administering to a subject an effective amount of a
substituted tetracycline compound of formula I: 1
[0006] wherein:
[0007] X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or
O;
[0008] R.sup.2, R.sup.2', R.sup.4', and R.sup.4" are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0009] R.sup.4 is NR.sup.4'R.sup.4", alkyl, alkenyl, alkynyl,
hydroxyl, halogen, or hydrogen;
[0010] R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety;
[0011] R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9
to form a ring;
[0012] R.sup.5 is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,
alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
alkyl carbonyloxy, or aryl carbonyloxy;
[0013] R.sup.6 and R.sup.6' are independently hydrogen, methylene,
absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl;
[0014] R.sup.7 is hydrogen, alkylamino, dialkylamino, or a malaria
interacting moiety;
[0015] R.sup.9 is hydrogen, or a malaria interacting moiety;
[0016] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl;
[0017] R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl;
[0018] Y' and Y are each independently hydrogen, halogen, hydroxyl,
cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; with the proviso that the compound of formula I is not
oxytetracycline, demeclocycline, doxycycline, chlorotetracycline,
minocycline, or tetracycline; and pharmaceutically acceptable salts
thereof.
[0019] This invention also relates, at least in part, to the use of
a substituted tetracycline compound of formula I in the preparation
of a medicament to treat or prevent malaria in a subject, e.g., a
mammal.
[0020] This invention pertains, at least in part, to a method for
treating or preventing malaria which is resistant to one or more
anti-malarial compounds such as, for example, proguanil,
chlorproguanil, trimethoprim, chloroquine, mefloquine,
lumefantrine, atovaquone, pyrimethamine-sulfadoxine,
pyrimethamine-dapsone, halofantrine, quinine, quinidine,
amodiaquine, amopyroquine, sulphonamides, artemisinin, arteflene,
artemether, artesunate, primaquine, and pyronaridine.
[0021] In another aspect, this invention also pertains to
pharmaceutical compositions which include an effective amount of
one of the above-described substituted tetracycline compounds and a
pharmaceutically acceptable carrier
[0022] This invention also features a packaged malarial treatment,
including one or more of the substituted tetracycline compounds of
the invention packaged with instructions for using the compound to
treat malaria.
DETAILED DESCRIPTION OF THE INVENTION
[0023] In one aspect, this invention pertains to methods of
treating or preventing malaria in a subject, by administering an
effective amount of a substituted tetracycline compound.
[0024] The term "malaria" includes the art recognized condition
known as "malaria" e.g., disorders which are caused by a protozoan
of the genus Plasmodium. Malaria is generally characterized by
symptoms such as headache, malaise, anemia, splenomegaly, and
paroxyms with cold, hot, and wet stages and is transmitted by
mosquitoes. (Winstanley (1998) Journal of the Royal College of
Physicians of London 32(3):203-207.) In a further embodiment, the
protozoan is selected from the group consisting of: P. falciparum,
P. vivax, P. ovale, and P. malariae.
[0025] The term "treated," "treating" or "treatment" includes the
diminishment or alleviation of at least one symptom associated or
caused by malaria, e.g., headache, malaise, anemia, splenomegaly,
and paroxyms with cold, hot, and wet stages. For example, treatment
can be diminishment of one or several symptoms of malaria or
complete eradication of malaria.
[0026] The term "prevented," or "preventing" includes
administration of a substituted tetracycline compound of the
invention to a subject who is not currently suffering from malaria,
such that the subject does not contract malaria for a period of
time after the administration and after exposure to malaria.
[0027] The term "tetracycline compounds" includes tetracycline
family members such as methacycline, sancycline, apicycline,
clomocycline, guamecycline, meglucycline, mepylcycline,
penimepicycline, pipacycline, etamocycline, penimocycline, etc. as
well as other tetracycline compounds having the characteristic
naphthacene A-B-C-D ring structure. Additional tetracycline
compounds can be found, for example, in U.S. patent application
Ser. No. 09/234,847, and U.S. Pat. Nos. 5,834,450; 5,532,227;
5,789,395; 5,639,742 and German patents DE 28 14 974 and DE 28 20
983. The entire contents of the aforementioned applications and
patents are hereby expressly incorporated herein by reference.
[0028] Recent research efforts have focused on developing new
tetracycline antibiotic compositions effective under varying
therapeutic conditions and routes of administration; and for
developing new tetracycline analogues which might prove to be equal
or more effective than the originally introduced tetracycline
families beginning in 1948. Representative of such developments
include U.S. Pat. Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331;
3,062,717; 3,557,280; 4,018,889; 4,024,272; 4,126,680; 3,454,697;
and 3,165,531. These issued patents are merely representative of
the range of diversity of investigations seeking tetracycline and
tetracycline analogue compositions which are pharmacologically
active, and the contents of each are expressly incorporated by
reference.
[0029] Historically, soon after their initial development and
introduction, the tetracyclines, regardless of specific formulation
or chemical structure, were found to be highly effective
pharmacologically against rickettsiae, a number of gram-positive
and gram-negative bacteria, and the agents responsible for
lymphogranuloma venereum, including conjunctivitis, and
psittacosis. Hence, tetracyclines became known as "broad spectrum"
antibiotics. With the subsequent establishment of their in vitro
antimicrobial activity, effectiveness in experimental infections,
and pharmacological properties, the tetracyclines as a class
rapidly became widely used for therapeutic purposes. However, this
widespread use of tetracyclines for both major and minor illnesses
and diseases led directly to the emergence of resistance to these
antibiotics even among highly susceptible bacterial species both
commensal and pathogenic (e.g., pneumococci and Salmonella). The
rise of tetracycline-resistant organisms has resulted in a general
decline in use of tetracyclines and tetracycline analogue
compositions as antibiotics of choice.
[0030] The terms "substituted tetracycline" and "substituted
tetracycline compounds" include tetracycline compounds of formula
I. In an embodiment, the term "substituted tetracycline compounds"
does not include oxytetracycline, demeclocycline, doxycycline,
chlorotetracycline, minocycline, and tetracycline. In a further
embodiment, "substituted tetracycline compounds" does not include
methacycline and sancycline. In another further embodiment, the
substituted tetracycline compounds of the invention do not include,
for example, compounds described in U.S. Pat. Nos. 6,043,231,
5,919,775, and 5,789,395, which are each incorporated in their
entirety herein by reference. The substituted tetracycline
compounds of the invention may be substituted such that certain
biological or physical properties are enhanced, e.g., such that the
substituted tetracycline compound is able to perform its intended
function, e.g., treat or prevent malaria.
[0031] In one embodiment, the substituted tetracycline compound of
the invention may have anti-microbial gram positive activity, as
measured by assays known in the art or the assay described in
Example 6. In an embodiment, the anti-microbial gram positive
activity of the substituted tetracycline compound is greater than
about 0.0001 .mu.g/ml, greater than about 0.05 .mu.g/ml, greater
than about 0.5 .mu.g/ml, greater than about 1.0 .mu.g/ml, or
greater than about 5.0 .mu.g/ml. Values and ranges included and/or
intermediate of the values set forth herein are also intended to be
within the scope of the present invention.
[0032] In another embodiment, the substituted tetracycline compound
of the invention has a cytotoxicity which allows the compound to be
administered in an effective amount to the subject with out causing
prohibitive cytotoxic side effects. In an embodiment, the
cytotoxicity of the substituted tetracycline compound of the
invention is greater than about 10 .mu.g/ml, about 15 .mu.g/ml,
about 20 .mu.g/ml, or about 25 .mu.g/ml as measured by cytoxicity
assays known in the art such as the assay described in Example
5.
[0033] In another embodiment, the substituted tetracycline compound
of the invention has a MIC which allows it to perform its intended
function, e.g., treat or prevent malaria in a subject. The MIC is a
measure of the concentration of the compound necessary to inhibit
the malaria parasite. The MIC can be tested using methods known in
the art as well as the in vitro method described in Example 3 or
the in vivo method described in Example 4. In an embodiment, the
MIC of a substituted tetracycline compound as measured in vitro is
about 1000 nM or less, about 900 nM or less, about 800 nM or less,
about 700 nM or less, about 600 nM or less, about 500 nM or less,
about 450 nM or less, about 400 nM or less, about 350 nM or less,
about 300 nM or less, about 250 nM or less, about 200 nM or less,
about 190 nM or less, about 180 nM or less, about 170 nM or less,
about 160 nM or less, about 150 nM or less, about 140 nM or less,
about 130 nM or less, about 120 nM or less, about 110 nM or less,
about 100 nM or less, about 90 nM or less, about 80 nM or less,
about 70 nM or less, about 60 nM or less, about 50 nM or less,
about 45 nM or less, about 40 nM or less, about 35 nM or less,
about 30 nM or less, about 25 nM or less, about 20 nM or less,
about 15 nM or less, about 12.5 nM or less, about 10 nM or less,
about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6
nM or less, about 5 nM or less, about 4.5 nM or less, about 4.0 nM
or less, about 3.5 nM or less, about 3.0 nM or less, about 2.5 nM
or less, about 2.0 nM or less, about 1.5 nM or less, about 1.0 nM
or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM
or less, about 0.2 nM or less, or about 0.11 nM or less.
[0034] In another embodiment, the MIC of a substituted tetracycline
compound as measured in vivo is about 500 mg/kg or less, about 250
mg/kg or less, about 200 mg/kg or less, about 190 mg/kg or less,
about 180 mg/kg or less, about 170 mg/kg or less, about 160 mg/kg
or less, about 150 mg/kg or less, about 140 mg/kg or less, about
130 mg/kg or less, about 120 mg/kg or less, about 110 mg/kg or
less, about 100 mg/kg or less, about 95 mg/kg or less, about 90
mg/kg or less, about 85 mg/kg or less, about 80 mg/kg or less,
about 75 mg/kg or less, about 70 mg/kg or less, about 65 mg/kg or
less, about 60 mg/kg or less, about 55 mg/kg or less, about 50
mg/kg or less, about 45 mg/kg or less, about 40 mg/kg or less,
about 35 mg/kg or less, about 30 mg/kg or less, about 29 mg/kg or
less, about 28 mg/kg or less, about 27 mg/kg or less, about 26
mg/kg or less, about 25 mg/kg or less, about 24 mg/kg or less,
about 23 mg/kg or less, about 22 mg/kg or less, about 21 mg/kg or
less, about 20 mg/kg or less, about 19 mg/kg or less, about 18
mg/kg or less, about 17 mg/kg or less, about 16 mg/kg or less,
about 15 mg/kg or less, 14 mg/kg or less, 13 mg/kg or less, 12
mg/kg or less, 11 mg/kg or less, 10 mg/kg or less, about 9 mg/kg or
less, about 8 mg/kg or less, about 7 mg/kg or less, about 6 mg/kg
or less, about 5 mg/kg or less, about 4.5 mg/kg or less, about 4
mg/kg or less, about 3.5 mg/kg or less, about 3 mg/kg or less,
about 2.5 mg/kg or less, about 2 mg/kg or less, a bout 1.5 mg/kg or
less, about 1 mg/kg or less, about 0.8 mg/kg or less, about 0.6
mg/kg or less, about 0.4 mg/kg or less, about 0.2 mg/kg or less,
about 0.1 mg/kg or less, about 0.05 mg/kg or less, or about 0.01
mg/kg or less.
[0035] This invention provides a method for treating or preventing
malaria in a subject by administering to the subject an effective
amount of a substituted tetracycline compound, such that malaria is
treated or prevented in said subject. The substituted tetracycline
compound is of formula I: 2
[0036] wherein:
[0037] X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or
O;
[0038] R.sup.2, R.sup.2', R.sup.4', and R.sup.4" are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0039] R.sup.4 is NR.sup.4'R.sup.4", alkyl, alkenyl, alkynyl,
hydroxyl, halogen, or hydrogen;
[0040] R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety;
[0041] R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9
to form a ring;
[0042] R.sup.5 is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,
alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
alkyl carbonyloxy, or aryl carbonyloxy;
[0043] R.sup.6 and R.sup.6' are independently hydrogen, methylene,
absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl;
[0044] R.sup.7 is hydrogen, alkylamino, dialkylamino, or a malaria
interacting moiety;
[0045] R.sup.9 is hydrogen, or a malaria interacting moiety;
[0046] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl;
[0047] R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl;
[0048] Y' and Y are each independently hydrogen, halogen, hydroxyl,
cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; with the proviso that the compound of formula I is not
oxytetracycline, demeclocycline, doxycycline, chlorotetracycline,
minocycline, or tetracycline; and pharmaceutically acceptable salts
thereof.
[0049] Examples of compounds of formula I which can be used in the
methods of the invention include substituted tetracycline compounds
wherein R.sup.2', R.sup.3, R.sup.8, R.sup.10, R.sup.11, and
R.sup.12 are hydrogen; R.sup.4 is NR'R" and R.sup.4' and R.sup.4"
are alkyl (e.g., methyl); and X is CR.sup.6R.sup.6'. The
substituted tetracycline compounds of the invention may also
include substituted minocycline derivatives, e.g., wherein R.sup.5,
R.sup.6, and R.sup.6 are hydrogen, and R.sup.7 is dialkylamino. The
invention also includes methods which use substituted doxycycline
derivatives, e.g., substituted tetracycline compounds of the
invention wherein R.sup.6 is alkyl, R.sup.6' is hydrogen, and
R.sup.7 is hydrogen. R.sup.5 may be hydroxyl or a prodrug moiety.
The invention also includes substituted sancycline compounds
wherein R.sup.5, R.sup.6, and R.sup.6' are hydrogen. In certain
embodiments, the substituted sancycline compounds include compounds
wherein at least one of R.sup.7 and R.sup.9 is a malaria
interacting moiety. In another embodiment, R.sup.4 is hydrogen.
[0050] In one embodiment, the substituted tetracycline compound of
the invention is substituted at least at the 7 or 9 position by a
substituent other than hydrogen (at either the 9 or 7 position) or
dimethyl amino at the 7 position.
[0051] In another embodiment, the substituted tetracycline compound
of the invention is substituted at the 7 or 9 position with a
malaria interacting moiety. The term "malaria interacting moiety"
is a moiety which allows the substituted tetracycline compound of
the invention to perform its intended function, e.g., treat or
prevent malaria. Not to be limited, but in an embodiment, the
malaria interacting moiety is a moiety which comprises from about 3
to 20 carbon, nitrogen, oxygen and sulfur atoms. The malaria
interacting moiety may further be substituted with hydrogen and
other substituents (e.g., halogens) which are not counted amongst
the 3 to 20 atoms. In a further embodiment, the malaria interacting
moiety comprises an aryl or heteroaryl moiety. Furthermore, the
aryl or heteroaryl moiety can be substituted with any substituent
which allows it to perform its intended function. The malaria
interacting moiety also may comprise alkenyl, alkynyl, and alkyl
moieties, which may also be substituted. In another embodiment, the
malaria interacting moiety comprises about 4 to 16 carbon, sulfur,
nitrogen, and oxygen atoms or from about 5 to about 15 carbon,
sulfur, nitrogen and oxygen atoms. Examples of malaria interacting
moieties include, but are not limited, to substituted and
unsubstituted aryl (e.g., substituted and unsubstituted phenyl),
alkyl, alkenyl, alkynyl, arylalkynyl, etc. In another embodiment,
the malaria interacting moiety is substituted aminoalkyl, e.g.,
alkylaminoalkyl, dialkylaminoalkyl, alkenylaminoalkyl,
alkynylaminoalkyl, aralkylaminoalkyl, arylaminoalkyl, etc.
[0052] In one embodiment, when R.sup.7 is a malaria interacting
moiety, the malaria interacting moiety may be halogen, thiol,
alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, arylalkyl, amino, arylalkenyl, arylalkynyl,
alkoxycarbonylalkylamino, or
--(CH.sub.2).sub.0-3NR.sup.7cC(.dbd.W')WR.sup.7a; wherein W is
CR.sup.7dR.sup.7e, NR.sup.7b, S, or O; W' is O or S; and R.sup.7a,
R.sup.7b, R.sup.7c, R.sup.7d, and R.sup.7e are each independently
hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety.
[0053] In one embodiment, when R.sup.9 is a malaria interacting
moiety, the malaria interacting moiety may be hydroxyl, halogen,
thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, arylalkyl, amino (e.g., unsubstituted
amino, alkylamino, dialkyl amino, alkoxycarbonylalkylamino, etc.),
arylalkenyl, arylalkynyl, or
--(CH.sub.2).sub.0-3NR.sup.9cC(=Z')ZR.sup.9a, wherein Z is
CR.sup.9dR.sup.9e, NR.sup.9b or O; Z' is O or S; and R.sup.9a,
R.sup.9b, R.sup.9c, R.sup.9d, and R.sup.9e are each independently
hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety. In embodiment, Z
is N and Z' is O, and R.sup.9a is optionally aryl. In another
embodiment, Z and Z' are O, and R.sup.9a is, for example, alkyl.
Further examples of R.sup.9 include --NR.sup.9cC(=Z')ZR.sup.9a,
wherein Z is CR.sup.9dR.sup.9e, NR.sup.9b or O; Z' is O or S; and
R.sup.9a, R.sup.9b, R.sup.9c, R.sup.9d, and R.sup.9e are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety. In another
embodiment, the malaria interacting moiety is substituted
aminoalkyl, e.g., alkylaminoalkyl, dialkylaminoalkyl,
aralkylaminoalkyl, alkenylaminoalkyl, alkynylaminoalkyl,
arylaminoalkyl, etc.
[0054] Examples of malaria interacting moieties include aryl groups
such as phenyl and heteroaryl groups (e.g., furanyl, imidazolyl,
benzothiophenyl, benzofuranyl, quinolinyl, isoquinolinyl,
pyridinyl, pyrazolyl, benzodioxazolyl, benzoxazolyl,
benzothiazolyl, benzoimidazolyl, methylenedioxyphenyl, indolyl,
thienyl, pyrimidyl, pyrazinyl, purinyl, pyrazolyl, oxazolyl,
isooxazolyl, naphthridinyl, thiazolyl, isothiazolyl, and
deazapurinyl). The aryl group may be substituted or unsubstituted.
Examples of substituents include, but are not limited, amino,
nitro, cyano, halogen (e.g., fluorine, chlorine, bromine, iodine,
etc.), hydroxy, thiol, formyl, acetyl, acyl, alkoxy (e.g.,
methylene dioxy, methoxy, ethoxy, propoxy, etc.) and heterocyclic
(e.g., morpholino, piperazine, etc.).
[0055] Other examples of malaria interacting moieties include
substituted and unsubstituted alkynyl groups. Examples of
substituted alkynyls include aryl alkynyls (e.g., a methoxy
substituted aryl alkynyl, cycloalkenyl substituted alkynyls, amino
substituted alkynyls, etc.). Other examples of malaria interacting
groups include substituted and unsubstituted alkenyl groups, such
as, for example, arylalkenyl groups. Furthermore, R.sup.9 groups
can also be substituted or unsubstituted alkyl groups (e.g., lower
alkyl groups, such as, for example, methyl, ethyl, propyl, butyl,
t-butyl, etc.). R.sup.9 may also be heterocyclic (e.g. thiazole,
amino thiazole, etc.), or substituted amino alkyl, amino
alkenyl.
[0056] The malaria interacting moiety may be substituted with one
or more substituents which allow it to performs its intended
function, e.g., treat or prevent malaria. Examples of substituents
include, but are not limited to, alkyl, alkenyl, alkynyl, halogen,
hydroxyl, alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,
arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl. In certain embodiments, the phenyl
is substituted with at least one alkyl, amino, heterocycle, alkoxy,
halogen, nitro, alkoxycarbonyl, dialkylamino, or alkylamino.
[0057] The methods of the invention also include the use of
substituted tetracycline compounds which are sancycline
derivatives, e.g., wherein R.sup.5, R.sup.6, and R.sup.6' are
hydrogen. Examples of sancycline derivatives include tetracycline
compounds wherein R.sup.7 is a malaria interacting moiety. Examples
of malaria interacting moieties which may be used for substituted
sancycline compounds of the invention include those described
above. Furthermore, other examples of malaria interacting moieties
include, but are not limited to, aryl group such as substituted or
unsubstituted phenyl or a heteroaryl moieties. Examples of
substituents include halogens (e.g., fluorine, chlorine, bromine,
iodine), alkoxy (e.g. methoxy, ethoxy, propoxy, methylene dioxy,
etc.), amino, and alkyl (e.g. methyl, ethyl, propyl, butyl,
t-butyl, etc.). In other embodiments, the substituted sancycline
compounds of the invention include compounds wherein R.sup.7 is
alkyl (e.g. methyl, ethyl, propyl, butyl, etc.), alkynyl (e.g. aryl
substituted, e.g., amino substituted arylalkynyl, etc.), halogen
(e.g., fluorine, chlorine, bromine, iodine), substituted or
unsubstituted methyl amido. Other substituted sancycline compounds
include compounds wherein R.sup.9 is hydrogen or a malaria
interacting moiety.
[0058] The methods of the invention also include methods which use
substituted doxycycline compounds as the substituted tetracycline
compound. Examples of substituted doxycycline compounds include
compounds wherein R.sup.5 is hydroxy or an ester groups, such as
alkyl esters (i.e., alkyl carbonyloxy groups, cyclohexane esters,
cycloheptane esters, pentyl esters, and ethyl esters).
[0059] Examples of the substituted tetracycline compounds of the
invention include the compounds shown in Table 1. Certain of the
substituted tetracycline compounds of the invention are shown
below: 3456789101112131415
[0060] Other substituted tetracycline compounds which maybe used in
the methods of the invention include, but are not limited to, the
compounds described in U.S.S. Nos. 60/346,930; 60/346,929;
60/347,065; 60/346,956; 60/367,048; 60/366,915; 60/367,045; Ser.
Nos. 09/823,884; 09/852,908; 09/882,505; 09/882,273; 09/894,805;
09/883,137; 09/895,797; 09/895,857; 09/895,812; 10/097,095 and
10/097,135; the contents of each of the aforementioned applications
are incorporated herein by reference in their entirety.
[0061] In a further embodiment, the substituted tetracycline
compounds are have a suitable oral bioavailability for the
treatment of malaria, e.g., after the substituted tetracycline
compounds are orally administered to the subject, the compounds are
able to perform their intended function, e.g., treat malaria.
Examples of methods which can be used to calculate the
bioavailability of a particular compound include methods known in
the art as well as the methods described in U.S.S. No. 60/318,580,
incorporated herein by reference.
[0062] In one embodiment, the substituted tetracycline compounds do
not include compounds which inhibit excess phospholipase A.sub.2
activity or production, as measured by the assay given in U.S. Pat.
No. 6,043,231. In another embodiment, the substituted tetracycline
compounds of the invention do not include compounds which inhibit
inducible nitric oxide synthase expression, as measured by the
assay given in U.S. Pat. No. 5,919,395. In another embodiment, the
substituted tetracycline compounds of the invention do not include
compounds which cause a decrease in the amount of nitric oxide
produced endogenously by a mammalian-system, as measured by the
method given in U.S. Pat. No. 5,789,395. Each of these three
patents are hereby incorporated herein by reference in their
entirety.
[0063] The term "subject" includes animals which are susceptible to
malaria, e.g. reptiles, birds, and mammals (e.g. dogs, cattle,
pigs, cats, horses, bears, sheep, mice, rats, rabbits, squirrels,
and most advantageously humans).
[0064] In a further embodiment, malaria for treatment using the
compositions and methods of the invention is resistant to one or
more anti-malarial compounds such as proguanil, chlorproguanil,
trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone,
pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine,
quinine, quinidine, amodiaquine, arnopyroquine, sulphonamides,
artemisinin, arteflene, artemether, artesunate, primaquine, and
pyronaridine.
[0065] The methods of the invention also include administering the
compounds of the invention in combination with a supplementary
compound. "Supplementary compounds" include anti-malarial compounds
and compounds that treat the symptoms of malaria. Supplementary
compounds may treat malaria directly, headache, malaise, anemia,
splenomegaly, and/or fever.
[0066] The term "in combination with" a supplementary compound is
intended to include simultaneous administration of the substituted
tetracycline compound and the supplementary compound,
administration of the substituted tetracycline compound first,
followed by the supplementary compound and administration of the
supplementary compound first, followed by the substituted
tetracycline compound.
[0067] For example, a "supplementary compound" can include
proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine,
lumefantrine, atovaquone, pyrimethamine-sulfadoxine,
pyrimethamine-dapsone, halofantrine, quinine, quinidine,
amodiaquine, amopyroquine, sulphonamides, artemisinin, arteflene,
artemether, artesunate, primaquine, pyronaridine, and
phosphatidylcholin synthesis inhibitors, such as G25
(1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide). Other
anti-malarial compounds not recited here can also be administered,
such as those which may be developed in the future or ones under
current investigation.
[0068] The invention also features a packaged malarial treatment,
including one or more substituted tetracycline compounds packaged
with instructions for using an effective amount of the compound to
treat malaria. In an embodiment, the substituted tetracycline
compound is not oxytetracycline, demeclocycline, doxycycline,
chlorotetracycline, minocycline, or tetracycline.
[0069] The substituted tetracycline compounds of the invention can
be synthesized using the methods described in Examples 1 and 2 and
in the following schemes. All novel substituted tetracycline
compounds described herein are included in the invention as
compounds. One of ordinary skill in the art will appreciate that
although the methods are illustrated generally for the synthesis of
7 substituted tetracycline compounds, similar procedures can be
used to generate the corresponding 9 position substituted
tetracycline compounds. Furthermore, although the schemes are
generally shown for one particular substituted tetracycline
compound (e.g., sancycline), the schemes and methods are generally
applicable to other substituted tetracycline compounds (e.g.,
tetracycline, minocycline, doxycycline, etc.). 16
[0070] 9- and 7-substituted tetracyclines can be synthesized by the
method shown in Scheme 1. As shown in Scheme 1, 9- and
7-substituted tetracycline compounds can be synthesized by treating
a tetracycline compound (e.g., doxycycline, 1A), with sulfuric acid
and sodium nitrate. The resulting product is a mixture of the
7-nitro and 9-nitro isomers (1B and 1C, respectively). The 7-nitro
(1B) and 9-nitro (1C) derivatives are treated by hydrogenation
using hydrogen gas and a platinum catalyst to yield amines 1D and
1E. The isomers are separated at this time by conventional methods.
To synthesize 7- or 9-substituted alkenyl derivatives, the 7- or
9-amino tetracycline compound (1E and 1F, respectively) is treated
with HONO, to yield the diazonium salt (1G and 1H). The salt (1G
and 1H) is treated with an appropriate halogenated reagent (e.g.,
R.sup.9Br, wherein R.sup.9 is an aryl, alkenyl, or alkynyl moiety)
to yield the desired compound (e.g., in Scheme 1,
7-cyclopent-1-enyl doxycycline (1H) and 9-cyclopent-1-enyl
doxycycline (1I)). 17
[0071] As shown in Scheme 2, substituted tetracycline compounds of
the invention wherein R.sup.7 is a carbamate or a urea derivative
can be synthesized using the following protocol. Sancycline (2A) is
treated with NaNO.sub.2 under acidic conditions forming 7-nitro
sancycline (2B) in a mixture of positional isomers.
7-nitrosancycline (2B) is then treated with H.sub.2 gas and a
platinum catalyst to form the 7-amino sancycline derivative (2C).
To form the urea derivative (2E), isocyanate (2D) is reacted with
the 7-amino sancycline derivative (2C). To form the carbamate (2G),
the appropriate acid chloride ester (2F) is reacted with 2C. 18
[0072] As shown in Scheme 3, substituted tetracycline compounds of
the invention, wherein R.sup.7 is a heterocyclic (i.e. thiazole)
substituted amino group can be synthesized using the above
protocol. 7-amino sancycline (3A) is reacted with
Fmoc-isothiocyanate (3B) to produce the protected thiourea (3C).
The protected thiourea (3C) is then deprotected yielding the active
sancycline thiourea (3D) compound. The sancycline thiourea (3D) is
reacted with an .alpha.-haloketone (3E) to produce a thiazole
substituted 7-amino sancycline (3F). 19
[0073] 7-alkenyl substituted tetracycline compounds, such as
7-alkynyl sancycline (4A) and 7-alkenyl sancycline (4B), can be
hydrogenated to form alkyl 7-substituted tetracycline compounds
(e.g., 7-alkyl sancycline, 4C). Scheme 4 depicts the selective
hydrogenation of the 7-position double or triple bond, in saturated
methanol and hydrochloric acid solution with a palladium/carbon
catalyst under pressure, to yield the product. 20
[0074] In Scheme 5, a general synthetic scheme for synthesizing
7-position aryl derivatives is shown. A Suzuki coupling of an aryl
boronic acid with an iodosancycline compound is shown. An iodo
sancycline compound (5B) can be synthesized from sancycline by
treating sancycline (5A) with at least one equivalent
N-iodosuccinimide (NIS) under acidic conditions. The reaction is
quenched, and the resulting 7-iodo sancycline (5B) can then be
purified using standard techniques known in the art. To form the
aryl derivative, 7-iodo sancycline (5B) is treated with an aqueous
base (e.g., Na.sub.2CO.sub.3) and an appropriate boronic acid (5C)
and under an inert atmosphere. The reaction is catalyzed with a
palladium catalyst (e.g., Pd(OAc).sub.2). The product (5D) can be
purified by methods known in the art (such as HPLC). Other 7-aryl
and alkynyl substituted tetracycline compounds can be synthesized
using similar protocols.
[0075] The 7-substituted tetracycline compounds of the invention
can also be synthesized using Stille cross couplings. Stille cross
couplings can be performed using an appropriate tin reagent (e.g.,
R--SnBu.sub.3) and a halogenated tetracycline compound, (e.g.,
7-iodosancycline). The tin reagent and the iodosancycline compound
can be treated with a palladium catalyst (e.g.,
Pd(PPh.sub.3).sub.2Cl.sub.2 or Pd(AsPh.sub.3).sub.2Cl.sub- .2) and,
optionally, with an additional copper salt, e.g., CuI. The
resulting compound can then be purified using techniques known in
the art. 21
[0076] The compounds of the invention can also be synthesized using
Heck-type cross coupling reactions. As shown in Scheme 6, Heck-type
cross-couplings can be performed by suspending a halogenated
tetracycline compound (e.g., 6-iodosancycline, 6A) and an
appropriate palladium or other transition metal catalyst (e.g.,
Pd(OAc).sub.2 and CuI) in an appropriate solvent (e.g., degassed
acetonitrile). The substrate, a reactive alkene (6B) or alkyne
(6D), and triethylamine are then added and the mixture is heated
for several hours, before being cooled to room temperature. The
resulting 7-substituted alkenyl (6C) or 7-substituted alkynyl (6E)
tetracycline compound can then be purified using techniques known
in the art. 22
[0077] To prepare 7-(2'-Chloro-alkenyl)-tetracycline compounds, the
appropriate 7-(alkynyl)-sancycline (7A) is dissolved in saturated
methanol and hydrochloric acid and stirred. The solvent is then
removed to yield the product (7B). 23
[0078] As depicted in Scheme 8, 5-esters of 9-substituted
tetracycline compounds can be formed by dissolving the
9-substituted compounds (8A) in strong acid (e.g. HF,
methanesulphonic acid, and trifluoromethanesulfonic acid) and
adding the appropriate carboxylic acid to yield the corresponding
esters (8B). 24
[0079] 13-substituted thiols can be synthesized by the method
outlined in Scheme 9, above. Generally, 13-substituted thiol ethers
(9B) can be synthesized by heating a tetracycline salt (9A) (such
as methacycline hydrochloride), AIBN (2,2'-azobisisobutyronitrile),
and a thiol in ethanol at reflux for six hours under an inert
atmosphere.
[0080] As shown in Scheme 10 below, 7 and 9 aminomethyl
tetracyclines may be synthesized using reagents such as
hydroxymethyl-carbamic acid benzyl ester. 25
[0081] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.),
branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl,
etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. The term alkyl
further includes alkyl groups, which can further include oxygen,
nitrogen, sulfur or phosphorous atoms replacing one or more carbons
of the hydrocarbon backbone. In an embodiment, a straight chain or
branched chain alkyl has 10 or fewer carbon atoms in its backbone
(e.g., C.sub.1-C.sub.10 for straight chain, C.sub.3-C.sub.10 for
branched chain), and more preferably 6 or fewer. Likewise,
preferred cycloalkyls have from 4-7 carbon atoms in their ring
structure, and more preferably have 5 or 6 carbons in the ring
structure.
[0082] Moreover, the term alkyl includes both "unsubstituted
alkyls" and "substituted alkyls", the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkenyl, alkynyl, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Cycloalkyls can be further substituted, e.g., with the substituents
described above. An "alkylaryl" or an "aralkyl" moiety is an alkyl
substituted with an aryl (e.g., phenylmethyl (benzyl)). The term
"alkyl" also includes the side chains of natural and unnatural
amino acids. Examples of halogenated alkyl groups include
fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,
dichloromethyl, trichloromethyl, perfluoromethyl, perchloromethyl,
perfluoroethyl, perchloroethyl, etc.
[0083] The term "aryl" includes groups, including 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, phenyl, pyrrole, furan,
thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine, and the like. Furthermore, the term "aryl" includes
multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g.,
naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,
isoquinoline, napthridine, indole, benzofuran, purine, benzofuran,
deazapurine, or indolizine. Those aryl groups having heteroatoms in
the ring structure may also be referred to as "aryl heterocycles",
"heterocycles," "heteroaryls" or "heteroaromatics". The aromatic
ring can be substituted at one or more ring positions with such
substituents as described above, as for example, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Aryl groups can also be fused or bridged with alicyclic or
heterocyclic rings which are not aromatic so as to form a polycycle
(e.g., tetralin, methylenedioxyphenyl).
[0084] The term "alkenyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double bond.
[0085] For example, the term "alkenyl" includes straight-chain
alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,
hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain
alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or
alkenyl substituted cycloalkenyl groups, and cycloalkyl or
cycloalkenyl substituted alkenyl groups. The term alkenyl further
includes alkenyl groups which include oxygen, nitrogen, sulfur or
phosphorous atoms replacing one or more carbons of the hydrocarbon
backbone. In certain embodiments, a straight chain or branched
chain alkenyl group has 6 or fewer carbon atoms in its backbone
(e.g., C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6 for
branched chain). Likewise, cycloalkenyl groups may have from 3-8
carbon atoms in their ring structure, and more preferably have 5 or
6 carbons in the ring structure. The term C.sub.2-C.sub.6 includes
alkenyl groups containing 2 to 6 carbon atoms.
[0086] Moreover, the term alkenyl includes both "unsubstituted
alkenyls" and "substituted alkenyls", the latter of which refers to
alkenyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0087] The term "alkynyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but which contain at least one triple bond.
[0088] For example, the term "alkynyl" includes straight-chain
alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl,
hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain
alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl
groups. The term alkynyl further includes alkynyl groups which
include oxygen, nitrogen, sulfur or phosphorous atoms replacing one
or more carbons of the hydrocarbon backbone. In certain
embodiments, a straight chain or branched chain alkynyl group has 6
or fewer carbon atoms in its backbone (e.g., C.sub.2-C.sub.6 for
straight chain, C.sub.3-C.sub.6 for branched chain). The term
C.sub.2-C.sub.6 includes alkynyl groups containing 2 to 6 carbon
atoms.
[0089] Moreover, the term alkynyl includes both "unsubstituted
alkynyls" and "substituted alkynyls", the latter of which refers to
alkynyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkyl groups, alkenyl groups, alkynyl groups,
halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0090] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to five carbon atoms in its backbone structure.
"Lower alkenyl" and "lower alkynyl" have chain lengths of, for
example, 2-5 carbon atoms.
[0091] The term "acyl" includes compounds and moieties which
contain the acyl radical (CH.sub.3CO--) or a carbonyl group. The
term "substituted acyl" includes acyl groups where one or more of
the hydrogen atoms are replaced by for example, alkyl groups,
alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0092] The term "acylamino" includes moieties where an acyl moiety
is bonded to an amino group. For example, the term includes
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido
groups.
[0093] The term "aroyl" includes compounds and moieties with an
aryl or heteroaromatic moiety bound to a carbonyl group. Examples
of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
[0094] The terms "alkoxyalkyl", "alkylaminoalkyl" and
"thioalkoxyalkyl" include alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or
sulfur atoms.
[0095] The term "alkoxy" includes substituted and unsubstituted
alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen
atom. Examples of alkoxy groups include methoxy, ethoxy,
isopropyloxy, propoxy, butoxy, and pentoxy groups and may include
cyclic groups such as cyclopentoxy. Examples of substituted alkoxy
groups include halogenated alkoxy groups. The alkoxy groups can be
substituted with groups such as alkenyl, alkynyl, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
[0096] The term "amine" or "amino" includes compounds where a
nitrogen atom is covalently bonded to at least one carbon or
heteroatom. The term "alkyl amino" includes groups and compounds
where the nitrogen is bound to at least one additional alkyl group.
The term "dialkyl amino" includes groups where the nitrogen atom is
bound to at least two additional alkyl groups. The term "arylamino"
and "diarylamino" include groups where the nitrogen is bound to at
least one or two aryl groups, respectively. The term
"alkylarylamino," "alkylaminoaryl" or "arylaminoalkyl" refers to an
amino group which is bound to at least one alkyl group and at least
one aryl group. The term "alkaminoalkyl" refers to an alkyl,
alkenyl, or alkynyl group bound to a nitrogen atom which is also
bound to an alkyl group.
[0097] The term "amide" or "aminocarboxy" includes compounds or
moieties which contain a nitrogen atom which is bound to the carbon
of a carbonyl or a thiocarbonyl group. The term includes
"alkaminocarboxy" groups which include alkyl, alkenyl, or alkynyl
groups bound to an amino group bound to a carboxy group. It
includes arylaminocarboxy groups which include aryl or heteroaryl
moieties bound to an amino group which is bound to the carbon of a
carbonyl or thiocarbonyl group. The terms "alkylaminocarboxy,"
"alkenylaminocarboxy," "alkynylaminocarboxy," and
"arylaminocarboxy" include moieties where alkyl, alkenyl, alkynyl
and aryl moieties, respectively, are bound to a nitrogen atom which
is in turn bound to the carbon of a carbonyl group.
[0098] The term "carbonyl" or "carboxy" includes compounds and
moieties which contain a carbon connected with a double bond to an
oxygen atom, and tautomeric forms thereof. Examples of moieties
which contain a carbonyl include aldehydes, ketones, carboxylic
acids, amides, esters, anhydrides, etc. The term "carboxy moiety"
or "carbonyl moiety" refers to groups such as "alkylcarbonyl"
groups where an alkyl group is covalently bound to a carbonyl
group, "alkenylcarbonyl" groups where an alkenyl group is
covalently bound to a carbonyl group, "alkynylcarbonyl" groups
where an alkynyl group is covalently bound to a carbonyl group,
"arylcarbonyl" groups where an aryl group is covalently attached to
the carbonyl group. Furthermore, the term also refers to groups
where one or more heteroatoms are covalently bonded to the carbonyl
moiety. For example, the term includes moieties such as, for
example, aminocarbonyl moieties, (where a nitrogen atom is bound to
the carbon of the carbonyl group, e.g., an amide), aminocarbonyloxy
moieties, where an oxygen and a nitrogen atom are both bond to the
carbon of the carbonyl group (e.g., also referred to as a
"carbamate"). Furthermore, aminocarbonylamino groups (e.g., ureas)
are also include as well as other combinations of carbonyl groups
bound to heteroatoms (e.g., nitrogen, oxygen, sulfur, etc. as well
as carbon atoms). Furthermore, the heteroatom can be further
substituted with one or more alkyl, alkenyl, alkynyl, aryl,
aralkyl, acyl, etc. moieties.
[0099] The term "urea" includes compounds that containing a
carbonyl group linked to two nitrogens. For example,
NH(C.dbd.O)NHAr is an aromatic urea group.
[0100] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom. The term "thiocarbonyl moiety" includes moieties
which are analogous to carbonyl moieties. For example,
"thiocarbonyl" moieties include aminothiocarbonyl, where an amino
group is bound to the carbon atom of the thiocarbonyl group,
furthermore other thiocarbonyl moieties include, oxythiocarbonyls
(oxygen bound to the carbon atom), aminothiocarbonylamino groups,
etc.
[0101] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl" which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0102] The term "ester" includes compounds and moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl,
alkenyl, or alkynyl groups are as defined above.
[0103] The term "thioether" includes compounds and moieties which
contain a sulfur atom bonded to two different carbon or hetero
atoms. Examples of thioethers include, but are not limited to
alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term
"alkthioalkyls" include compounds with an alkyl, alkenyl, or
alkynyl group bonded to a sulfur atom which is bonded to an alkyl
group. Similarly, the term "alkthioalkenyls" and alkthioalkynyls"
refer to compounds or moieties where an alkyl, alkenyl, or alkynyl
group is bonded to a sulfur atom which is covalently bonded to an
alkynyl group.
[0104] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-.
[0105] The term "halogen" includes fluorine, bromine, chlorine,
iodine, etc. The term "perhalogenated" generally refers to a moiety
where all hydrogens are replaced by halogen atoms.
[0106] The terms "polycyclyl" or "polycyclic" include moieties with
two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls,
aryls and/or heterocyclyls) in which two or more carbons are common
to two adjoining rings, e.g., the rings are "fused rings". Rings
that are joined through non-adjacent atoms are termed "bridged"
rings. Each of the rings of the polycycle can be substituted with
such substituents as described above, as for example, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
alkylaminoacarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or
an aromatic or heteroaromatic moiety.
[0107] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0108] The term "heterocycle" or "heterocyclic" includes saturated,
unsaturated, aromatic ("heteroaryls" or "heteroaromatic") and
polycyclic rings which contain one or more heteroatoms. Examples of
heterocycles include, for example, benzodioxazole, benzofuran,
benzoimidazole, benzothiazole, benzothiophene, benzoxazole,
deazapurine, furan, indole, indolizine, imidazole, isooxazole,
isoquinoline, isothiaozole, methylenedioxyphenyl, napthridine,
oxazole, purine, pyrazine, pyrazole, pyridazine, pyridine,
pyrimidine, pyrrole, quinoline, tetrazole, thiazole, thiophene, and
triazole. Other heterocycles include morpholine, piprazine,
piperidine, thiomorpholine, and thioazolidine. The heterocycles may
be substituted or unsubstituted. Examples of substituents include,
for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, alkylaminoacarbonyl, aralkylaminocarbonyl,
alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,
alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate, phosphonato, phosphinato, cyano, amino (including alkyl
amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0109] It will be noted that the structure of some of the compounds
of this invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(e.g., all enantiomers and diastereomers) are included within the
scope of this invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by classical separation
techniques and by stereochemically controlled synthesis.
Furthermore, the structures and other compounds and moieties
discussed in this application also include all tautomers thereof.
The term "prodrug moiety" includes moieties which can be
metabolized in vivo to a hydroxyl group and moieties which may
advantageously remain esterified in vivo. Preferably, the prodrugs
moieties are metabolized in vivo by esterases or by other
mechanisms to hydroxyl groups or other advantageous groups.
Examples of prodrugs and their uses are well known in the art (See,
e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19). The prodrugs can be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form or hydroxyl
with a suitable esterifying agent. Hydroxyl groups can be converted
into esters via treatment with a carboxylic acid. Examples of
prodrug moieties include substituted and unsubstituted, branch or
unbranched lower alkyl ester moieties, (e.g., propionoic acid
esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl
esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl
esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters
(e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester),
aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g.,
with methyl, halo, or methoxy substituents) aryl and aryl-lower
alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides,
and hydroxy amides. Preferred prodrug moieties are propionoic acid
esters and acyl esters.
[0110] In another aspect, this invention further pertains to a
pharmaceutical composition which includes an effective amount of a
substituted tetracycline compound to treat malaria in a subject and
a pharmaceutically acceptable carrier.
[0111] This invention also pertains to the use of a compound of
formula I in the preparation of medicament to treat or prevent
malaria in a subject.
[0112] The pharmaceutical composition may also include a
supplementary compound.
[0113] "Supplementary compounds" include anti-malarial compounds
and compounds that treat the symptoms of malaria. Supplementary
compounds may treat malaria directly, headache, malaise, anemia,
splenomegaly, and/or fever. Examples of supplementary anti-malarial
compounds include proguanil, chlorproguanil, trimethoprim,
chloroquine, mefloquine, lumefantrine, atovaquone,
pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine,
quinine, quinidine, amodiaquine, amopyroquine, sulphonamides,
artemisinin, arteflene, artemether, artesunate, primaquine,
pyronaridine, and combinations thereof.
[0114] The language "pharmaceutically acceptable carrier" includes
substances capable of being co-administered with the substituted
tetracycline compound(s), and which allow the substituted
tetracycline compound to perform its intended function, e.g., treat
or prevent malaria. Examples of such carriers include solutions,
solvents, dispersion media, delay agents, emulsions and the like.
The use of such media for pharmaceutically active substances are
well known in the art. Any other conventional carrier suitable for
use with the substituted tetracycline compounds of the present
invention are included.
[0115] For example, one or more compounds of the invention may be
administered alone to a subject, or more typically a compound of
the invention will be administered as part of a pharmaceutical
composition in mixture with conventional excipient, i.e.,
pharmaceutically acceptable organic or inorganic carrier substances
suitable for parenteral, oral or other desired administration and
which do not deleteriously react with the active compounds and are
not deleterious to the recipient thereof. Suitable pharmaceutically
acceptable carriers include but are not limited to water, salt
solutions, alcohol, vegetable oils, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
petroethral fatty acid esters, hydroxymethylcellulose,
polyvinylpyrrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously react with the active compounds.
[0116] Some of the substituted tetracycline compounds of the
invention suitably may be administered to a subject in a protonated
and water-soluble form, e.g., as a pharmaceutically acceptable salt
of an organic or inorganic acid, e.g., hydrochloride, sulfate,
hemi-sulfate, phosphate, nitrate, acetate, oxalate, citrate,
maleate, mesylate, etc. Also, where an appropriate acidic group is
present on a compound of the invention, a pharmaceutically
acceptable salt of an organic or inorganic base can be employed
such as an ammonium salt, or salt of an organic amine, or a salt of
an alkali metal or alkaline earth metal such as a potassium,
calcium or sodium salt.
[0117] The substituted tetracycline compounds can be administered
to a subject in accordance with the invention by any of a variety
of routes such as topical (including transdermal, buccal or
sublingual), and parenteral (including intraperitoneal,
subcutaneous, intravenous, intradermal or intramuscular injection).
In one embodiment, the substituted tetracycline compounds are
administered orally.
[0118] For parenteral application, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Therapeutic compounds will be formulated in sterile form in
multiple or single dose formats such as being dispersed in a fluid
carrier such as sterile physiological saline or 5% saline dextrose
solutions commonly used with injectables.
[0119] For enteral or oral administration, particularly suitable
are tablets, dragees or capsules having talc and/or carbohydrate
carrier binder or the like, the carrier preferably being lactose
and/or corn starch and/or potato starch. A syrup, elixir or the
like can be used where a sweetened vehicle is employed. Sustained
release compositions can be formulated including those where the
active component is protected with differentially degradable
coatings, e.g., by microencapsulation, multiple coatings, etc.
[0120] For topical applications, the substituted tetracycline
compound(s) can be suitably admixed in a pharmacologically inert
topical carrier such as a gel, an ointment, a lotion or a cream.
Such topical carriers include water, glycerol, alcohol, propylene
glycol, fatty alcohols, triglycerides, fatty acid esters, or
mineral oils. Other possible topical carriers are liquid
petrolatum, isopropylpalmitate, polyethylene glycol, ethanol 95%,
polyoxyethylene monolauriate 5% in water, sodium lauryl sulfate 5%
in water, and the like. In addition, materials such as
anti-oxidants, humectants, viscosity stabilizers and the like also
may be added if desired.
[0121] The actual preferred amounts of active compounds used in a
given therapy will vary according to the specific compound being
utilized, the particular compositions formulated, the mode of
application, the particular site of administration, etc. Optimal
administration rates for a given protocol of administration can be
readily ascertained by those skilled in the art using conventional
dosage determination tests conducted with regard to the foregoing
guidelines.
[0122] In general, compounds of the invention for treatment can be
administered to a subject in dosages used in prior tetracycline
therapies. See, for example, the Physicians' Desk Reference. For
example, a suitable effective dose of one or more compounds of the
invention will be in the range of from 0.01 to 100 milligrams per
kilogram of body weight of recipient per day, preferably in the
range of from 0.1 to 50 milligrams per kilogram body weight of
recipient per day, more preferably in the range of 1 to 20
milligrams per kilogram body weight of recipient per day. The
desired dose is suitably administered once daily, or several
sub-doses, e.g. 2 to 5 sub-doses, are administered at appropriate
intervals through the day, or other appropriate schedule.
[0123] For purposes of the comparison the doses other particular
tetracycline related compounds are summarized. The adult dose for
tetracycline, oxytetrcycline, and chlortetracycline is generally
250 mg every 6 hours by mouth with 500 mg in serious infections.
For children under 50 kg, doxycycline 4 mg/kg is generally given on
the first day with 2 mg/kg in subsequent days. For intramuscular
tetracycline, the appropriate adult dose is generally 100 mg 2 to 3
times daily. For intravenous/intrapleural tetracycline, the usually
adult dose is generally 500 mg twice daily.
[0124] It will also be understood that normal, conventionally known
precautions will be taken regarding the administration of
tetracyclines generally to ensure their efficacy under normal use
circumstances. Especially when employed for therapeutic treatment
of humans and animals in vivo, the practitioner should take all
sensible precautions to avoid conventionally known contradictions
and toxic effects. Thus, the conventionally recognized adverse
reactions of gastrointestinal distress and inflammations, the renal
toxicity, hypersensitivity reactions, changes in blood, and
impairment of absorption through aluminum, calcium, and magnesium
ions should be duly considered in the conventional manner.
[0125] The language "effective amount" of the substituted
tetracycline compound is that amount necessary or sufficient to
control malaria in a subject, e.g., to prevent or ameliorate the
various morphological and somatic symptoms of malaria. The
effective amount can vary depending on such factors as the size and
weight of the subject, the type of illness, or the particular
substituted tetracycline compound. For example, the choice of the
substituted tetracycline compound can affect what constitutes an
"effective amount". One of ordinary skill in the art would be able
to study the aforementioned factors and make the determination
regarding the effective amount of the substituted tetracycline
compound without undue experimentation. An in vivo assay also can
be used to determine an "effective amount" of a substituted
tetracycline compound. The ordinarily skilled artisan would select
an appropriate amount of a substituted tetracycline compound for
use in the aforementioned in vivo assay. Preferably, the effective
amount of the substituted tetracycline compound is effective to
treat a subject, e.g., human, suffering from malaria.
[0126] The term "subject" includes animals which are capable of
having malaria. Examples of subject include, but are not limited
to, birds (i.e. geese, ducks), reptiles, ruminants (e.g., cattle
and goats), mice, rats, hamsters, dogs, cats, horses, pigs, sheep,
lions, tigers, bears, monkeys, chimpanzees, and, in a preferred
embodiment, humans.
[0127] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
[0128] Exemplification of the Invention:
[0129] Compounds of the invention may be made as described below,
with modifications to the procedure below within the skill of those
of ordinary skill in the art.
EXAMPLE 1
Synthesis of 7-Substituted Tetracyclines
[0130] 7 Iodo Sancycline
[0131] One gram of sancycline was dissolved in 25 mL of TFA
(trifluoroacetic acid) that was cooled to 0 C (on ice). 1.2
equivalents of N-iodosuccinimide (NIS) was added to the reaction
mixture and reacted for forty minutes. The reaction was removed
from the ice bath and was allowed to react at room temperature for
an additional five hours. The mixture was then analyzed by HPLC and
TLC, was driven to completion by the stepwise addition of NIS.
After completion of the reaction, the TFA was removed in vacuo and
3 mL of MeOH was added to dissolve the residue. The methanolic
solution was the added slowly to a rapidly stirring solution of
diethyl ether to form a greenish brown precipitate. The 7-iodo
isomer of sancycline was purified by treating the 7-iodo product
with activated charcoal., filtering through Celite, and subsequent
removal of the solvent in vacuo to produce the 7-isomer compound as
a pure yellow solid in 75% yield.
[0132] MS(M+H) (formic acid solvent) 541.3.
[0133] .backslash.R.sup.t: Hypersil C18 BDS Column, 11.73
[0134] .sup.1H NMR (Methanol d.sub.4-300 MHz) .delta. 7.87-7.90 (d,
1H), 6.66-6.69 (d, 1H), 4.06 (s, 1H), 2.98 (s, 6H), 2.42 (m, 1H),
2.19 (m, 1H), 1.62 (m, 4H), 0.99 (m, 2H)
[0135] 7-Phenyl Sancycline
[0136] 7-iodosancycline, 150 mg (0.28 mM), Pd(OAc).sub.2 and 10 mL
of MeOH are added to a flask with a stir bar and the system
degassed 3.times. using argon. Na.sub.2CO.sub.3 (87 mg, 0.8 mM)
dissolved in water and argon degassed is added via syringe is added
along with phenylboronic acid (68 mg, 0.55 mM) in MeOH that was
also degassed. The reaction was followed by HPLC for 2 hours and
cooled to room temperature. The solution was filtered, and dried to
produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18 reverse-phase silica. The fraction at 36-38 minutes was
isolated, and the solvent removed in vacuo to yield the product
plus salts. The salts were removed by extraction into 50:25:25
water, butanol, ethyl acetate and dried in vacuo. This solid was
dissolved in MeOH and the HCl salt made by bubbling in HCl gas. The
solvent was removed to produce the product in 42% yield as a yellow
solid.
[0137] Rt 21.6 min: MS (M+H, formic acid solvent): 491.3
[0138] .sup.1H NMR (Methanol d.sub.4-300 MHz) .delta. 7.87 (d,
J=8.86 Hz, 1H), 7.38 (m, 5H), 6.64 (d, 8.87 Hz, 1H), 4.00 (s, 1H),
3.84 (s, 2H), 3.01 (s, 6H), 2.46 (m, 2H), 1.63 (m, 4H), 0.95 (m,
2H)
[0139] 7-(4'-Chlorophenyl) Sancycline
[0140] 7-iodosancycline, 500 mg (0.91 mM), Pd(OAc).sub.2 21 mg, and
20 mL of MeOH are added to a flask with a stir bar and the system
degassed 3.times.using argon. Na.sub.2CO.sub.3 (293 mg, 2.8 mM)
dissolved in water and argon degassed is added via syringe is added
along with 4-Cl-phenylboronic acid (289 mg, 1.85 mM) in MeOH that
was also degassed. The reaction was followed by HPLC for 45 minutes
and cooled to room temperature. The solution was filtered, and
dried to produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18reverse-phase silica. The fraction at 39 minutes was isolated,
and the solvent removed in vacuo to yield the product plus salts.
The salts were removed by extraction into 50:25:25 water, butanol,
ethyl acetate and dried in vacuo. This solid was dissolved in MEOH
and the HCl salt made by bubbling in HCl gas. The solvent was
removed to produce the product in 57% yield as a yellow solid.
[0141] Rt 20.3 min: MS (M+H, formic acid solvent): 525.7
[0142] .sup.1H NMR (Methanol d14-300 MHz) .delta. 7.49-7.52 (d,
J=8.54 Hz, 11H), 6.99-7.01 (d, 8.61 Hz, 1H), 4.12 (s, 1H), 3.67 (m,
1H), 3.06 (s, 6H), 2.58 (m, 2H), 1.62(m, 4H), 1.01 (m, 2H)
[0143] 7-(4'-Fluorophenyl) Sancycline
[0144] 7-iodosancycline, 200 mg (0.3 mM), Pd(OAC).sub.2 8.3 mg, and
10 mL of MeOH are added to a flask with a stir bar and the system
degassed 3.times.using argon. Na.sub.2CO.sub.3 (104 mg, 1.11 mM)
dissolved in water and argon degassed is added via syringe is added
along with 4-F-phenylboronic acid (104 mg, 0.7 mM) in MeOH that was
also degassed. The reaction was followed by HPLC for 20 minutes and
cooled to room temperature. The solution was filtered, and dried to
produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18 reverse-phase silica. The fraction at 19-20 minutes was
isolated, and the solvent removed in vacuo to yield the product
plus salts. The salts were removed by extraction into 50:25:25
water, butanol, ethyl acetate and dried in vacuo. This solid was
dissolved in MeOH and the HCl salt made by bubbling in HCl gas. The
solvent was removed to produce the product in 47% yield as a yellow
solid.
[0145] Rt 19.5 min: MS (M+H, formic acid solvent): 509.4
[0146] .sup.1H NMR (Methanol d14-300 MHz) .delta. 6.92-6.95 (d,
1H), 7.45-7.48 (d, 1H), 7.15-7.35 (m, 4H), 4.05 (s, 1H), 3.62 (m,
1H), 3.08 (s, 6U), 2.55 (m, 2H), 1.65(m, 4H), 1.00 (m, 2H)
[0147] 7-(4'-Iodo-1',3'-carboethoxy-1',3'-butadiene) Sancycline
[0148] 7-I-Sancycline (1 gm, 1.86 mmol), was dissolved in 25 mL of
acetonitrile and was degassed and purged with nitrogen (three
times). To this suspension Pd(OAC).sub.2 (20 mg, 0.089 mmol), CuI
(110 mg, 0.053)mmol), (o-tolyl).sub.3P (56 mg, 0.183 mmol) were
added and purged with nitrogen. Ethyl propiolate (1 mL) and
triethylamine (1 mL) were added to the suspension. It turned to a
brown solution upon addition of Et.sub.3N. The reaction mixture was
then heated to 70 degrees C. for two hours. Progress of the
reaction was monitored by HPLC. It was then cooled down to room
temperature and was filtered through celite. Evaporation of the
solvent gave a brown solid, which was then purified on preparative
HPLC to give a yellow solid.
[0149] 7-(2'-Chloroethenyl)-Sancycline
[0150] To a solution/suspension of 0.65 g (1 mmol) of 7-iodo
sancycline, 0.05 g tetrakis triphenyl phosphinato palladate, 0.012
g palladium acetate, 0.05 g copper (I) iodide in 10 mL
acetonitrile, 2 mL triethylamine and 0.5 g trimethylsilyl acetylene
was added at room temperature. The reaction proceeded for two hours
before being filtered through a celite bed and concentrated. The
crude product was purified by preparative HPLC. The collected
fractions were concentrated and the residue was taken up in about 1
mL of methanol and 2 mL of HCl saturated methanol. The product was
precipitated with ether. The solids were filtered off and dried
under reduced pressure. NMR spectroscopy and LC-MS showed that the
compound was 7-(2-chloroethenyl) sancycline.
[0151] 7-(4'-aminophenyl) Sancycline
[0152] To a solution of 200 mg of 7-(4-nitrophenyl) sancycline in
50 mL methanol, 10 mg of 10% palladium on charcoal catalyst was
added. The reaction mixture was shaken under 40 psi hydrogen
pressure for 2 hours and was then filtered followed by
concentration. The residue was further purified by preparative
HPLC. 35 mg was isolated as the HCl salt and the structure was
proved by NMR and LC-MS to be
[0153] 7-(4-aminophenyl) sancycline. 26
[0154] 7-1-Sancycline (1 gm, 1.86 mmol), taken in 25 mL of
acetonitrile was degassed and purged with nitrogen (three times).
To this suspension Pd(OAc).sub.2 (20 mg, 0.089 mmol), CuT (10 mg,
0.053 mmol), (o-tolyl).sub.3P (56 mg, 0.183 mmol) were added and
purged with nitrogen for few minutes. NN-Dimethylpropyne (308 mg,
3.72 mmol) and triethylamine (1 mL) were added to the suspension.
It was turned into a brown solution upon addition of Et.sub.3N. The
reaction mixture was then heated to 70.degree. C. for 3 hours.
Progress of the reaction was monitored by HPLC. It was then cooled
down to room temperature and was filtered through celite.
Evaporation of the solvent gave a brown solid, which was then
purified on preparative HPLC to give a yellow solid. The structure
of this compound has been characterized using 1H NMR, HPLC, and
MS.
[0155] 7-(2'-Chloro-3-Hydroxypropenyl)-Sancycline 27
[0156] 7-(alkynyl)-sancycline (100 mg) was taken in 20 ml of
saturated MeOH/HCl and stirred for 20 min. The solvent was then
evaporated to give a yellow powder. The structure of this compound
has been characterized using 1H NMR, HPLC, and MS.
[0157] 7-(3'-Methoxyphenylethyl)-Sancycline 28
[0158] 7-(3'-Methoxyphenylethynyl)-sancycline (1 mmol) was taken in
saturated solution of MeOH/HCl. To this solution 10% Pd/C was added
and was subjected to hydrogenation at 50 psi for 12 hrs. It was
then filtered through celite. The solvent was evaporated to give a
yellow powder. Finally, it was precipitated from MeOH/diethylether.
The structure of this compound has been characterized using 1H NMR,
HPLC, and MS.
[0159] (2-Dimethylamino-Acetylamino)-Sancycline 29
[0160] NN-Dimethylglycine (1.2 mmol) was dissolved in DMF (5 mL)
and O-Benzotriazol-1-yl-N,N,N,N,-tetramethyluronium
hexafluorophosphate (HBTU, 1.2 mmol) was added. The solution was
then stirred for 5 minutes at room temperature. To this solution,
7-aminosancycline (1 mmol) was added, followed by the addition of
diisopropylethyl amine (DIEA, 1.2 mmol). The reaction was then
stirred at room temperature for 2 hours. The solvent, DMF, was
removed on vacuum. The crude material was dissolved in 5 mL of MeOH
and filtered using autovials and purified using preparative HPLC.
The structure of the product has been characterized using 1H NMR,
HPLC, and MS.
[0161] 7-(N-Methylsulphonamidopropargylamine) Sancycline 30
[0162] To a mixture of 7-iodosancycline mono trifluoroacetic acid
salt (1 g; 1.53 mmoles), palladium II acetate(17.2 mg; 0.076
mmoles), tetrakis triphenylphosphine palladium (176.8 mg; 0.153
mmoles), and copper (I) iodide(49 mg; 0,228 mmoles) was added 15 ml
of reagent grade acetonitrile in a clean dry 2 necked round bottom
flask. The reaction was purged with a slow steam of argon gas, with
stirring, for 5 minutes before the addition (in one portion as a
solid) of N-methylsulphonamidopropargyl amine. The sulphonamide was
prepared by a method known in the art (J. Med. Chem 31(3) 1988;
577-82). This was followed by one milliliter of triethylamine (1
ml; 0.726 mg; 7.175 mmoles) and the reaction was stirred, under an
argon atmosphere, for approximately 1.0 hour at ambient
temperature. The reaction mixture was suctioned filtered through a
pad of diatomaceous earth and washed with acetonitrile. The
filtrates were reduced to dryness under vacuo and the residue was
treated with a dilute solution of trifluroroacetic acid in
acetonitrile to adjust the pH to approximately 2. The residue was
treated with more dilute trifluoroacetic acid in acetonitrile,
resulting in the formation of a precipitate, which was removed via
suction filtration. The crude filtrates were purified utilizing
reverse phase HPLC with DVB as the solid phase; and a gradient of
1:1 methanol/acetonitrile 1% trifluoroacetic acid and 1%
trifluoroacetic acid in water. The appropriate fractions were
reduced to dryness under reduced pressure and solid collected. The
product was characterized via .sup.1H NMR, mass spectrogram and LC
reverse phase.
[0163] 7-(2'-methoxy-5'-formylphenyl)sancycline 31
[0164] 7-iodo-sancycline (Ig, 1.53 mmol), Pd(OAc).sub.2 (34 mg,
0.153 mmol), and MeOH (50 mL) were combined in a 250 mL 2 neck
round bottom flask equipped with a condenser and argon line. The
solution was then purged with argon (15 min) while heated in an oil
bath to approximately 70.degree. C. Sodium carbonate (482 mg, 4.58
mmol) was dissolved in water (3-5 mL) and added to reaction flask.
The flask was then purged with argon for another 5 minutes.
2-Methoxy-5-formylphenyl boronic acid (333 mg, 1.83 mmol) was
dissolved in MeOH (5 mL) and added to reaction flask. The flask was
then purged again with argon for 10 minutes. The reaction was
monitored to completion within 3 hours. The contents of the flask
were filtered through filter paper and the remaining solvent was
evacuated. To make the hydrochloric acid salt, the residue was
dissolved in MeOH (sat. HCl) to make the HCl salt. The solution was
then filtered and the solvent was evacuated. The product was then
characterized by .sup.1H NMR, LC-MS.
[0165] 7-(2'-Methoxy-5'-N,N'-Dimethylaminomethylphenyl)Sancycline
32
[0166] 7-(2'-methoxy-5'-formylphenyl)sancycline (1 g, 1.82 mmol),
dimethylamine HCl (297 mg, 3.64 mmol), triethylamine (506 .mu.L,
3.64 mmol), and 1,2-DCE (7 mL) were combined in a 40 mL vial. The
contents were dissolved within several minutes of shaking or
stirring. Sodium triacetoxyborqhydride (772 mg, 3.64 mmol) was then
added as a solid. The reaction was monitored by HPLC and LC-MS and
was complete within 3 hours. The reaction was quenched with MeOH
(20 mL) and the solvent was subsequently evacuated. The residue was
redissolved in 3 mL DMF and separated on a C-18 column. Fractions
from the prep column dried down in-vacuo and the HCl salt was made
by dissolving contents in methanol (sat. HCl). The solvent was
reduced and a yellow powder obtained. Characterized by .sup.1H NMR,
LC-MS, HPLC.
[0167] 7-Furanyl Sancycline
[0168] 7-iodo sancycline (1.3 mg) and Pd(OAc).sub.2 were taken in
100 mL of methanol and purged with argon for five minutes at
70.degree. C. To this solution was added a solution of sodium
carbonate (44 mg) in water (previously purged with argon). A yellow
precipitate was obtained and the mixture was heated for another ten
minutes. 3-Furanyl boronic acid (333 mg, solution in DMF, purged
with argon) was then added and the mixture was heated for another
two hours at 70.degree. C. The reaction was monitored by MPLC/MS.
When the reaction was complete, the mixture was filtered through
celite and the solvent was removed to give a crude material. The
crude material was purified by precipitating it with ether (200
ml). The yellow precipitate was filtered and purified using
preparative HPLC. The hydrochloride salt was made by dissolving the
material in MeOH/HCl and evaporating to dryness. The identity of
the resulting solid was confirmed using HPLC, MS, and NMR.
EXAMPLE 2
Preparation of 9-Substituted Minocyclines
[0169] Preparation of 9-Iodominocycline
[0170] To 200 ml of 97% methanesulfonic acid was slowly added, at
ambient temperature, portionwise [30 g;56.56 mM] of
minocycline-bis-hydrochloride salt. The dark yellow brown solution
was then stirred at ambient temperature while [38 g;169.7 mM] of
N-iodosuccinimide was added, in six equal portions, over 3.0 hours
time. The reaction was monitored via analytical LC, noting the
disappearance of the starting material.
[0171] The reaction was slowly quenched into 2L of ice cold water
containing [17.88 g; 1134.1 mM] of sodium thiosulfate with rapid
stirring. This quench was stirred for approximately 30 minutes at
ambient temperature. The aqueous layer was then extracted with
6.times.200 ml of ethyl acetate before the aqueous was poured onto
[259.8 g;3.08M] of sodium hydrogen carbonate containing 300 ml of
n-butanol. The phases were split and the aqueous extracted with
4.times.250 ml of n-butanol. The organic fractions were combined
and washed with 3.times.250 ml of water and once with 250 ml of
saturated brine. The resulting organic phase was reduced to dryness
under reduced pressure. The residue was suspended in methanol
(.about.600 ml) and anhydrous HCl gas was bubbled into this mixture
until solution occurred This solution was reduced to dryness under
reduced pressure. The filtrates were reduced to dryness under
reduced pressure. The resulting material was triturated with 300 ml
of methyl t-butyl ether and isolated via filtration. This material
was redissolved in 300 ml of methanol and treated with 0.5 g of
wood carbon, filtered and filtrates reduced to dryness under
reduced pressure. The material was again powdered under methyl
t-butyl ether, isolated via suction filtration and washed with more
ether, and finally hexanes. The material was vacuum dried to give
22.6 g of a light yellow brown powder.
[0172] General Procedure For Preparation of 9-Alkynyl Minocycline
Compounds
[0173] 1 mmol 9-iodo minocycline, 50 mg tetrakis
triphenylphosphinato palladate, 12 mg palladium acetate, 32 mg
copper (I) iodide are dissolved/suspended in 10 ml acetonitrile. 2
to 5 ml triethylamine and 3 to 5 mmol alkynyl derivative is added.
The reaction mixture is vigorously stirred between ambient
temperature to 70.degree. C. The reaction time is 2-24 hours. When
the reaction is completed the dark suspension is filtered through a
celite bed and concentrated. The crude product is purified by prep
HPLC. The combined fractions are concentrated and taken up in
.about.1 ml methanol. .about.3 ml HCl saturated methanol is added,
and the product is precipitated with ether.
[0174] General Procedure For Preparation of 9-Aryl Minocycline
Compounds
[0175] 0.15 mmol of 9-iodominocycline, PdOAc (3.2 mg), 229 .mu.l 2M
Na.sub.2CO.sub.3 and 2 equivalents of phenyl boronic acid were
dissolved/suspended in 10 ml methanol. The reaction flask was
purged with argon and the reaction run for a minimum of four hours
or until HPLC monitoring shows consumption of starting material
and/or the appearance of products. The suspension was filtered
through celite, and subject to purification by prep HPLC on a
divinylbenzene or CIE reverse-phase column.
[0176] 9-(4-Trifluoromethoxyphenylureido)-Methyl Minocycline 33
[0177] To 3 mL of dimethylformamide was added 150 mg (0.25 mmol) of
9-methyl aminominocyline trihydrochloride and 67 mL (0.50 mmol) of
triethylamine at 25.degree. C. With stirring, 75 mL (0.50 mmol) of
4-trifluoromethoxyphenylisocyanate was added and the resulting
reaction mixture was stirred at 25.degree. C. for two hours. The
reaction was monitored by analytical HPLC (4.6.times.50 mm reversed
phase Luna C18 column, 5 minute linear gradient 1-100% B buffer, A
buffer was water with 0.1% trifluoroacetic acid, B buffer was
acetonitrile with 0.1% trifluoroacetic acid). Upon completion, the
reaction was quenched with 1 mL of water and the pH adjusted to
approximately 2.0 with concentrated HCl. The solution was filtered
and the compound purified by preparative HPLC. The product yield
was 64 mg (37% yield). The purity of the product was 95%, as
determined by LCMS (M+1=690).
[0178] 9-(4'Carboxy phenyl) Minocycline 34
[0179] In a clean, dry reaction vessel, was placed
9-iodominocycline [500 mg; 0.762 mmoles]bis HCl salt, palladium
(II) acetate [17.2 mg; 0.076 mmoles] along with 10 ml of reagent
grade methanol. The solution was immediately purged, with stirring,
with a stream of argon gas for approximately 5 minutes. The
reaction vessel was brought to reflux and to it was sequentially
added via syringe 2M potassium carbonate solution [1.91 ml; 3.81
mmoles], followed by a solution of p-carboxyphenyl boronic acid
[238.3 mg; 1.53 mmoles]in 5 ml of reagent DMF. Both of these
solutions were previously degassed with argon gas for approximately
5 minutes. The reaction was heated for 45 minutes, the progress was
monitored via reverse phase HPLC. The reaction was suctioned
filtered through a pad of diatomaceous earth and washed with DMF.
The filtrates were reduced to an oil under vacuum and residue
treated with t-butylmethyl ether. Crude material was purified via
reverse phase HPLC on DVB utilizing a gradient of water and
methanol/acetonitrile containing 1.0% trifluoroacetic acid. Product
confirmed by mass spectrum: found M+1 578.58; the structure
corroborated with 1H NMR.
EXAMPLE 3
Assessment of Antimalarial Activity in vitro
[0180] The following protocol is adapted from Antimicrob. Agents
Chemother. 40:1600-1603, 1996 and was used in the instant
examples.
[0181] Preparation of parasites: Strains of P. falciparum were
grown continuously in culture. A 6% suspension of human type A+
erythrocytes were prepared in culture medium which consists of
powdered RPMI 1640 diluted in sterile water with 25 mM HEPES, 32 mM
NaHCO3 and 10% heat-inactivated human type A+ fresh frozen plasma
(in acid-citrate-dextrose anticoagulant). Stock cultures were
maintained in 5 mL of the 6% erythrocyte suspension in 25 mL tissue
culture flasks. The flasks were flushed with a gas mixture of 5%
CO.sub.2, 5%O.sub.2 and 90% N.sub.2. The flasks were then sealed
and incubated at 37.degree. C. The cultures were maintained so that
less than 2% of the erythrocytes were infected at any one time. For
experiments, samples of the stock cultures were diluted in culture
medium containing sufficient noninfected type A+ human erythrocytes
to yield a final hematocrit of 1.5% and parasitemia of 0.25 to 0.5%
in preparation of addition to the microtiter plates.
[0182] Preparation of drugs: All compounds were dissolved initially
in DMSO at a stock concentration of 20 mg/mL. The final dilution
contained less than 1% DMSO which has no measurable effect on the
parasites in this system.
[0183] Microtiter plate setup: 25 .mu.l of the culture medium was
placed in each well of a 96 well microtiter plate. 25 .mu.l of the
DMSO drug solution was added to two separate wells of the plate.
After the drugs were added to the wells, an automatic diluter was
used to make serial twofold dilutions. A constant volume (200
.mu.l) of the parasitized erythrocyte suspension was added to each
well of the microtiter plate except for the controls. The control
were treated with 200 .mu.l of an equivalent suspension of
nonparasitized type A human erythrocytes. The total volume in every
well was 225 .mu.l. After preparation, the plates were placed in a
humidified airtight box with a mixture of 5% O.sub.2, 5% CO.sub.2
and 90% N.sub.2, sealed and placed in an incubator at 30.degree. C.
for 24 to 48 hours.
[0184] Harvesting parasites and scintillation counting: At the end
of the second incubation period, each plate was harvested using a
automated cell harvester. The instrument essentially aspirates and
deposits the particulate contents of each of the wells onto small
disks of filter paper which are then thoroughly washed with
distilled water. Each disk is then counted using a scintillation
counter.
[0185] Table 1, which follows, shows the relative MIC values
obtained for certain substituted tetracycline compounds of the
invention. * represents good inhibition of parasite growth, **
represents very good inhibition of parasite growth, *** represent
extremely good inhibition of parasite growth. MIC represents the
minimum concentration of the compound that inhibits P. falciparum
growth after incubation at 30.degree. C. for 24 to 48 hours.
1TABLE 1 Mol Tox- Ac- ID STRUCTURE M + H Weight icity tivity A 35
444.4402 *** B 36 457.4822 ** *** C 37 442.4244 ** *** D 38
444.4402 ** *** E 39 414.414 * *** F 40 464.8585 *** G 41 460.4396
** *** H 42 473.4 472.4648 ** I 43 543.6 542.5846 * ** J 44 546.5
545.545 ** K 45 515.5 514.531 * ** L 46 555.6 554.5956 * ** M 47
516.6 515.562 ** N 48 557.5 456.4542 ** O 49 460.4 459.4548 ** P 50
527 526.542 * ** Q 51 487.5 486.4774 ** R 52 575.6 574.6446 ** S 53
640.5 639.7442 * T 54 527.6 526.5852 * *** U 55 555.4 554.6388 ** V
56 501.3 500.5042 * ** W 57 545.4 544.6184 * ** X 58 513.5 512.5152
* Y 59 499.3 498.4884 * Z 60 569.4 568.6224 ** AA 61 684.8 683.8152
* * AB 62 529 528.601 ** AC 63 671.3 670.733 * AD 64 595 594.5063
** AE 65 597.707 * AF 66 543 542.5414 * AG 67 602 601.609 * AH 68
561 560.6178 ** AI 69 528.5146 ** AJ 70 529 520.5378 ** AK 71
546.5756 * ** AL 72 510.5426 * AM 73 551 550.564 * ** AN 74 557
556.6114 ** ** AO 75 657.7192 * AP 76 560 559.6176 * ** AQ 77 514
513.5894 ** AR 78 872.1092 * AS 79 573.5956 * ** AT 80 747.7596 *
AU 81 518.522 * AV 82 640 639.7036 * AW 83 537 536.5125 * AX 84
681.0208 * ** AY 85 534 533.5798 ** AZ 86 694.0628 ** BA 87
577.6081 * ** BB 88 564.5661 * ** BC 89 641.6792 ** * BD 90 588
587.6254 ** BE 91 564.5476 * ** BF 92 607.3 806.6712 * *** BG 93
592.0041 * BH 94 667 666.2072 * ** BI 95 681 680.4922 ** BJ 96
509.3 508.5021 * ** BK 97 533.3 532.5488 ** BL 98 588.3 587.4122 **
BM 99 501.4 500.5474 * BN 100 563.3 562.575 ** BO 101 567.3
566.6246 * BP 102 482.2 481.5042 * BQ 103 572.6138 * * BR 104 494.1
493.3101 * ** BS 105 535.5092 * *** BT 106 540.2 539.602 * ** BU
107 564.6 563.606 * ** BV 108 589.3 588.6338 ** BW 109 607.3
606.6243 * ** BX 110 549.3 548.5482 * ** BY 111 579.3 578.5774 * *
BZ 112 578.3 577.5896 ** CA 113 729.4 728.73446 ** * CB 114 699.4
698.70826 ** * CC 115 686.4 685.66626 ** CD 116 716.3 715.69246 **
CE 117 534.3 533.5798 * ** CF 118 527.3 526.4826 * *** CG 119 535.2
534.5214 * ** CH 120 587.2 586.5203 * ** CI 121 562.3 561.5902 **
*** CJ 122 497.2 496.5338 * ** CK 123 569.2 568.0249 ** CL 124
545.3 544.6034 ** CM 125 535.3 534.5646 * *** CN 126 549.3 548.5944
** CO 127 558.5896 ** CP 128 577.5896 * ** CQ 129 603.3 602.47 * *
CR 130 570.3 569.5608 * ** CS 131 570.3 569.5608 * * CT 132 558.3
557.6018 * ** CU 133 481.2 480.5164 * ** CV 134 588.3 587.628 * **
CW 135 576.2 575.5923 * ** CX 136 574.2 573.6012 * * CY 137 496.2
495.531 ** * CZ 138 521.2 520.581 * ** DA 139 521.2 520.581 * ** DB
140 607.1 606.5019 * ** DC 141 572.3 571.6286 * *** DD 142 496.3
495.531 ** *** DE 143 496.3 495.531 *** DF 144 506.3 505.5262 **
*** DG 145 562.3 561.6334 * ** DH 146 533.2 579.6486 ** ** DI 147
533.2 532.5488 * *** DJ 148 556.2 558.5099 * ** DK 149 505.3
504.5384 * ** DL 150 578.3 577.5896 ** * DM 151 481.2 480.5164 * **
DN 152 535.2 534.5646 * ** DO 153 560.2 559.5312 * ** DP 154 512.2
511.5304 ** *** DQ 155 580.2 579.5622 * * DS 156 568.2 567.5265 **
* DT 157 599.3 596.611 ** * DU 158 564.2 563.5628 ** * DV 159 543.3
533.5798 * ** DW 160 475.2 474.8969 * ** DX 161 533.3 532.5488 * **
DY 162 563.3 562.575 * ** DZ 163 531.2 530.9789 * ** EA 164 522.3
521.5256 * *** EB 165 531.3 530.533 * ** EC 166 521.3 520.5378 * **
ED 167 517.3 516.5494 * ** EE 168 534.5646 * ** EF 169 544.5598 *
** EG 170 537.2 536.5804 ** ** EH 171 737.2 736.513 ** * EI 172
532.2 531.564 * *** EJ 173 539.4 538.5992 ** * EK 174 551.2 550.564
* ** EL 175 603.3 602.5994 * * EM 176 559.3 558.5896 ** * EN 177
443.2 442.4876 * ** EO 178 506.3 506.5262 * ** EP 179 506.2
504.9231 ** ** EQ 180 519.2 518.5652 * ** ER 181 555.3 555.0261 *
** ET 182 547.3 546.5756 * * EU 183 529.3 528.5604 * ** EV 184
511.3 510.5606 * ** EW 185 547.3 546.6188 * ** EX 186 555.3
554.9829 * ** EY 187 516.3 515.5214 * ** EZ 188 525.3 524.9567 * **
FA 189 549.2 548.5482 * *** FB 190 511.3 510.5606 * ** FC 191 483.3
482.489 ** ** FD 192 530.3 529.5482 * *** FE 193 516.2 515.5214 *
** FF 194 519.2 518.522 * ** FG 195 591.2 590.6316 ** * FH 196
483.3 482.4458 ** * FI 197 457.3 456.4512 ** *** FJ 198 533.2
532.5241 * ** FK 199 549.3 548.5914 * ** FL 200 545.3 544.5598 * **
FM 201 549.3 548.5914 * ** FN 202 567.1 595.4455 * *** FO 203 495.3
494.5432 * ** FP 204 513.2 512.6016 * ** FQ 205 615.3 614.6536 *
*** FR 206 529.5 528.5604 * ** FS 207 531.3 530.5762 * ** FT 208
515.3 514.6496 * ** FU 209 500.3 499.5626 ** *** FV 210 574.2
573.6444 ** *** FW 211 610.3 609.6774 * *** FX 212 548.4 547.6066
** *** FY 213 620.3 619.67 ** *** FZ 214 556.3 565.5834 ** ** GA
215 526.2 525.514 ** * GB 216 592.3 591.6194 ** *** GC 217 605.3
604.6584 ** ** GD 218 620.3 619.6298 ** ** GE 219 575.3 577.6328 **
*** GF 220 646.4 645.754 ** * GG 221 535.3 524.6126 * ** GH 222
577.3 576.6048 ** ** GI 223 540.3 539.584 ** ** GJ 224 499.3
498.5748 * ** GK 225 588.3 587.628 * ** GL 226 573.3 572.6164 ** *
GM 227 559.3 558.5896 * * GN 228 665.2 664.7288 ** ** GO 229 671.3
671.0621 * ** GP 230 610.3 609.6099 ** *** GQ 231 593.4 592.6638 **
* GR 232 617.3 616.6848 ** * GS 233 617.4 616.7126 ** * GT 234
596.2 569.0353 *** GU 235 563.2 563.0059 ** GV 236 609.2 608.6648 *
GW 237 627.2 627.0493 * GX 238 544.5602 ** GY 239 571.3 570.598 **
GZ 240 775.4 774.80586 * ** HA 241 514.534 ** *** HB 242 499.2
498.4884 ** * HC 243 632.5 631.7242 ** *** HD 244 606.5 605.6864 **
*** HE 245 592.3 591.6596 ** *** HF 246 604.4 603.6706 ** *** HG
247 616.3 617.5974 ** *** HH 248 590.3 589.687 ** HI 249 534.3
533.5366 ** *** HJ 250 514.3 513.5462 ** HK 251 601.4 600.6674 * HL
252 559.3 558.587 ** * HM 253 486.3 485.4925 *** HN 254 486.3
485.4926 *** HO 255 564.3 563.609 * HP 256 571.4 570.6442 * HQ 257
569.3 568.6686 * * HR 258 589.3 588.6132 ** ** HS 259 648.3
647.6834 ** ** HT 260 544.3 543.5724 ** * HU 261 620.2 619.6268 **
*** HV 262 520.2 519.553 ** *** HW 263 521.2 520.5378 ** *** HX 264
644.3 633.6968 ** * HY 265 564.2 563.5628 ** *** HZ 266 481.1
480.4732 * *** IA 267 486.3 485.5358 ** * IB 268 542.2 541.5998 **
** IC 269 549.2 548.5944 ** ** ID 270 525.2 524.486 ** * IE 271
458.2 457.4822 ** ** IF 272 527.28 526.5882 ** ** IG 273 572.4
571.629 ** ** IH 274 584.4 582.6832 ** ** II 275 585.6218 ** *** IJ
276 557.4 556.6576 ** *** IK 277 594.4 593.6352 ** * IL 278 584.3
583.64 ** ** IM 279 570.3 569.6132 ** * IN 280 572.3 571.629 ** **
IO 281 516.3 515.5188 ** * IP 282 500.3 499.5194 ** * IQ 283 514.3
513.503 ** *** IR 284 642.3 641.6946 ** * IS 285 613.4 612.7048 **
***
EXAMPLE 4
Assessment of Antimalarial Activity in vivo
[0186] The assessment is performed with P. vinckei, a murine
parasite that consistently causes a rapidly fatal malaria, and is
an excellent model for drug efficacy. However, other murine
parasites which are available (e.g. P. berghei) can also be studied
using similar methodology.
[0187] 20 gm Swiss Webster mice are inoculated intraperitoneally
with 10.sup.6 P. vinckei-infected erythrocytes obtained from
another infected mouse. Twelve hours after infection, treatment is
initiated by the intraperitoneal injection of test compounds.
Treatment is continued twice-a-day (BID) for four days. The
progress of malaria infections in experimental and control
(injected with diluent only) mice is followed by daily examinations
of blood smears obtained from tail veins. The pharmacological
endpoint is parasitemia >50%. Uninfected animals are followed
for 6 weeks, and the animals that remain uninfected through this
period are considered long-term cures.
[0188] The test compounds are injected into the stomach of the test
mice by gavage. A number of variations of standard in vivo protocol
may be utilized for specific purposes. For example, dosing
intervals may be altered based on the known pharmacokinetics or
observed initial efficacy data for a compound. Protocols may also
be altered to more closely mimic true treatment (with delay of
therapy after inoculation of parasites) or chemoprophylaxis (with
treatment before the inoculation of parasites) conditions.
[0189] For all in vivo experiments, the mice are monitored daily,
for at least the first two weeks of an experiment, with blood
smears. Counts per 1000 erythrocytes provide parasitemias, and the
parasitemias are then plotted over time, and results for control
and experimental animals are compared.
EXAMPLE 5
Mammalian Cytotoxicity Assay
[0190] COS-1 and CHO cell suspensions are prepared, seeded into
96-well tissue culture treated black-walled microtiter plates
(density determined by cell line), and incubated overnight at
37.degree. C., in 5% CO.sub.2 and approximately 95% humidity. The
following day serial dilutions of drug are prepared under sterile
conditions and transferred to cell plates. Cell/Drug plates are
incubated under the above conditions for 24 hours. Following the
incubation period, media/drug is aspirated and 50 .mu.l of
Resazurin is added. Plates are then incubated under the above
conditions for 2 hours and then in the dark at room temperature for
an additional 30 minutes. Fluorescence measurements are taken
(excitation 535 nm, emission 590 nm). The IC.sub.50 (concentration
of drug causing 50% growth inhibition) is then calculated. The
cytotoxicity of both unsubstituted minocycline and doxycycline were
found to be greater than 25. Substituted tetracycline compounds
with good cytotoxicities are indicated with * in Table 1.
Substituted tetracycline compounds with very good cytotoxicities
are indicated with ** in Table 1.
EXAMPLE 6
In vitro Anti-Bacterial Activity Assay
[0191] The following assay is used to determine the efficacy of the
tetracycline compounds against common bacteria. 2 mg of each
compound is dissolved in 100 ill of DMSO. The solution is then
added to cation-adjusted Mueller Hinton broth (CAMHB), which
results in a final compound concentration of 200 .mu.g per ml. The
tetracycline compound solutions are diluted to 50 .mu.L volumes,
with a test compound concentration of 0.098 .mu.g/ml. Optical
density (OD) determinations are made from fresh log-phase broth
cultures of the test strains. Dilutions are made to achieve a final
cell density of 1.times.10.sup.6 CFU/ml. At OD=1, cell densities
for different genera should be approximately:
2 E. coli 1 .times. 10.sup.9 CFU/ml S. aureus 5 .times. 10.sup.8
CFU/ml Enterococcus sp. 2.5 .times. 10.sup.9 CFU/ml
[0192] 50 .mu.l of the cell suspensions are added to each well of
microtiter plates. The final cell density should be approximately
5.times.10.sup.5 CFU/ml. These plates are incubated at 35.degree.
C. in an ambient air incubator for approximately 18 hr. The plates
are read with a microplate reader and are visually inspected when
necessary. The MIC is defined as the lowest concentration of the
tetracycline compound that inhibits growth.
[0193] Equivalents
[0194] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
[0195] The contents of all references, patents, and patent
applications cited throughout this application are hereby
incorporated by reference. The appropriate components, processes,
and methods of those patents, applications and other documents may
be selected for the present invention and embodiments thereof.
* * * * *